AU2020220242B2 - Compositions and methods for enhanced lymphocyte-mediated immunotherapy - Google Patents

Abstract

Lymphocytes having a suppressed autophagy gene useful in immunotherapy are disclosed. The lymphocytes can express an antigen targeting receptor such as a chimeric antigen receptor (CAR) or endogenous or engineered T-cell receptor to target cells expressing a tumor-specific antigen. Methods of making and using such lymphocytes are disclosed. Some such lymphocytes are useful in conducting CAR-T or TCR-T therapy.

Description

Editorial Note 2020220242 Description pages are non- sequential. Page 7C is missing filed amendments has pages 7, 7A, 7B, 7D, and is followed by page 8. Their is no page 7C

COMPOSITIONS AND METHODS FOR ENHANCED LYMPHOCYTE-MEDIATED IMMUNOTHERAPY

Reference to Related Applications

[0001] This application claims priority to, and the benefit of, United States provisional patent application No. 62/804658 filed 12 February 2019, the entirety of which is incorporated by reference herein in its entirety for all purposes

Government License Rights

[0002] This invention was made, in part, with government support under Grant Number OC170169 made under Award Number W81XWH-18-1-0264 awarded by the U.S. Army Medical Research and Development Command. The government has certain rights in the invention.

Field of the Invention

[0003] Some embodiments of this invention relate to genetically engineered lymphocytes having improved efficacy as immunotherapeutics. Some embodiments of this invention relate to methods and compositions to modify the metabolism of lymphocytes in order to improve their efficacy as immunotherapeutics. Some embodiments relate to methods for the genetic manipulation of autophagy in chimeric antigen receptor T-cells (CAR-T) or engineered or endogenous T cell receptor T-cells (TCR-T) to enhance their effectiveness for immunotherapy of cancer.

Background

[0004] CAR-T-cells are engineered with an antigen-binding domain such as an antibody single chain variable fragment (scFv) to bind antigens expressed on the cell surface of tumor cells. In standard therapy, a CAR construct is transduced into autologous CD3+ T cells, expanded ex vivo and then infused into a patient. "New generation" CAR-T-cells contain optimized CD3( signalling domains fused in combination with co-stimulatory molecules such as CD28, 41BBL and CD27 (3). Despite these modifications, the persistence of CAR-T-cells in solid tumors remains poor. Thus, other barriers besides pro survival signals control the function of CAR-T-cells in the tumor microenvironment.

[0005] Despite decades of research, treatments for ovarian cancer have not improved patient outcomes. High-grade serous carcinoma (HGSC), the most common histotype, is often diagnosed at stage 3-4 disease and patients receive debulking surgery followed by several rounds of chemotherapy. The 5-year survival rate for stage 4 disease is < 20%. Although vaccines, checkpoint blockade and T-cell therapy have been explored, early trials have not improved overall patient survival. However, there is unequivocal evidence that the presence and function of tumor infiltrating lymphocytes (TIL) is strongly associated with improved survival implying that the immune system is beneficial in this disease (4).

[0006] Folate receptors (FR) exist as 3 isoforms, a, P, and y, and function in folic acid and folate uptake. High levels of aFR have been observed in ovarian, breast and lung cancers but are low in normal tissue (5). In one trial, 14 ovarian cancer patients who received infusions of aFR-CAR-T-cells with or without IL-2 showed no evidence of clinical response despite treatment being well-tolerated. Using radiolabel tracer imaging, the authors concluded that the aFR-CAR-T-cells were unable to persist shortly after infusion (2). Therefore, the current approaches to use aFR-CAR-T in ovarian cancer have not been successful, in part, due to the lack of persistence of cells after infusion. However, this work provides an established safety profile for aFR-CAR-T therapy.

[0007] Manufacturing CAR-T-cells requires specialized infrastructure, patients must undergo lymphodepletion prior to T-cell infusions and acute life-threatening immune adverse events can arise. Moreover, CAR-T-cells have shown limited efficacy in solid tumors (6). One possibility to explain the lack of observed efficacy for solid tumors is a metabolic barrier imposed by the tumor ecosystem due to the high metabolic demands of rapidly proliferating tumor cells. Ultimately, this may cause a loss of T-cell function and persistence. Thus, it appears that metabolism can significantly impact T-cell behaviour providing a unique opportunity to modify T-cell metabolism and achieve better therapeutic success in solid cancers.

[0008] Deregulation of central metabolism is a universal hallmark of cancers, an observation reported for ovarian cancer (7). There is widespread appreciation that cancers use glucose and glutamine at high rates to serve as the main biosynthetic precursor for cell growth and proliferation (8). Recent work demonstrates that T-cells have adaptive metabolism depending on their state of activation and differentiation (7,9-12). For example, the transition from a naive T-cell to an activated effector cell is accompanied by switch from oxidative metabolism to a more glycolytic phenotype (9). The metabolic similarities of proliferating T-cells and tumor cells is striking and implies that insufficiencies in glucose and other nutrients that are essential to support proliferation can contribute to reduced T-cell function and exhaustion (10).

[0009] Metabolic competition could suppress T-cells that have been infused as part of T-cell therapy. One report found that CD8+ tumor infiltrating lymphocytes (TIL) in murine tumors were more functional in tumors with reduced glucose consumption compared to tumors with high rates of glycolysis suggesting that glucose consumption by tumors directly impaired T cell activity (10). This defect was rescued by expressing metabolic enzymes that restore T cell glycolysis. In another report, loss of mitochondrial mass in tumor-infiltrating lymphocytes (TIL) was associated with a reduction in type 1 cytokines, increased checkpoint inhibitor expression and loss of anti-tumor activity (13). However, enforced expression of PGC1a, a transcription factor involved in mitochondria biogenesis, restored anti-tumor immunity. In the face of metabolic stress, reprogramming TIL to use alternative fuels may help sustain their anti-tumor activity. Despite these studies, there are no reports examining the role of metabolism on human TIL known to the inventors.

[0010] Autophagy is a form of catabolic metabolism where cells engulf portions of the cytosol and degrade cellular contents in the lysosome for metabolite recycling, protein quality control or destruction of damaged organelles (14). Autophagy is largely a survival pathway activated by nutrient and growth factor deprivation (14). T-cells lacking autophagy genes such as Atg5 or Atg7 have impaired thymocyte development and a reduction in peripheral T-cells (15). In line with this, it has been reported that autophagy is essential for CD8+ effector T-cell survival and memory development (16, 17).

[0011] Given the fundamental role that metabolism plays in organismal homeostasis and cancer, one might expect deleterious or pro-oncogenic phenotypes associated with manipulating the autophagy pathway. In the case of autophagy deficiency, haploinsufficency of one autophagy gene, Beclin-1, has been found to promote tumor formation and monoallelic deletion is observed in 40-75% of sporadic breast, ovarian and prostate cancer (18). Moreover, aged Atg5 or Atg7 liver-specific knockouts develop spontaneous liver tumors (19). It is believed that loss of Atg5 in T-cells does not lead to oncogenesis based on the inventors' own observations. Moreover, gain- or loss-of function in metabolic genes is required but not sufficient for tumorigenesis. For instance, T-cell specific Glut1 transgenic mice do not spontaneously develop tumors (20).

[0012] There have been reports using CRISPR/Cas9 for gene-editing in primary human T-cells (21, 22). A Chinese group has successfully knocked out PD-1 in T-cells, though the details of their strategy have yet to be reported. Another group was able to target the CD19 CAR to the T-cell receptor-a locus and cause enhanced tumor rejection in a mouse model (22). Most recently, a group at the University of Pennsylvania led by Carl June successfully conducted a first-in-human phase 1 trial testing the safety and feasibility of multiplex CRISPR-Cas9 editing in T-cells (24).

[0013] Many types of cells are involved in killing tumor cells, including NK cells; T-cells including CD34+, CD4+ or CD8+ T-cells, Treg cells, tissue-resident memory T cells (TRM cells), natural killer T-cells (NKT); B-cells, and the like. These cells act through a similar mechanism of recognizing an antigen such as a tumor-specific antigen on the surface of the tumor cell and acting to kill such cell.

[0014] Some embodiments of the present invention address an unmet need in the field for improved treatments using lymphocytes for immunotherapy, including CAR-T therapy, especially for treatment of solid cancers, for example those of the ovary, breast or lung.

[0015] The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

Summary

[0016] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above described problems have been reduced or eliminated, while other embodiments are directed to other improvements, or at least provide the public with a useful choice.

[0017] In one aspect, a lymphocyte having an antigen targeting receptor and a suppressed autophagy gene is provided. In some aspects, the lymphocyte is provided with a nucleic acid encoding the antigen targeting receptor inserted within a locus of the autophagy gene to disrupt expression of the autophagy gene. In some aspects, the autophagy gene is located at a first locus of the genome and the nucleic acid encoding the antigen targeting receptor is inserted at a second locus of the genome that is different from the first locus. In some such aspects, the autophagy gene is knocked out or disrupted at the first locus or suppressed in some other manner, for example using RNAi.

[0018] In one aspect, a method of conducting immunotherapy is provided that involves administering to a subject an engineered lymphocyte as described in this specification. In one aspect, a method of conducting immunotherapy is provided in which lymphocytes that have been modified to suppress an autophagy gene are administered to a subject. In some aspects, a method of conducting immunotherapy is provided in which lymphocytes that have been modified to both suppress an autophagy gene and express an antigen targeting receptor are administered to a subject. In some aspects, the immunotherapy is used to treat cancer.

[0019] In one aspect, a method of making a lymphocyte for use in immunotherapy is provided and involves modifying the lymphocyte to suppress an autophagy gene. In some such aspects, the lymphocyte is further modified to express a desired antigen targeting receptor.

[0020] In some aspects, any suitable technique may be used to suppress expression of the autophagy gene, including using CRISPR-Cas, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), Sleeping Beauty (SB), RNAi, meganucleases, or megaTALs. In some aspects, the autophagy gene is disrupted or knocked out, for example using CRISPR-Cas, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), Sleeping Beauty (SB), meganucleases, or megaTALs.

[0021] In some aspects, methods and compositions to improve the anti-tumor efficacy of lymphocytes are provided. In some aspects, methods and compositions to improve the anti-tumor efficacy of cytotoxic lymphocytes are provided. In some aspects, methods and compositions to improve the anti-tumor efficacy of cytotoxic cells are provided. In some aspects, methods and compositions to improve the anti-tumor efficacy of lymphocytes including NK cells; T-cells including CD34+, CD4+ or CD8+ T-cells, Treg cells, tissue-resident memory T cells (TRM cells), natural killer T-cells (NKT); B-cells; or the like are provided.

[0022] In some aspects, the genetically engineered lymphocytes express an antigen targeting receptor such as a chimeric antigen receptor (CAR) or an endogenous or engineered T-cell receptor which targets a desired tumor-specific antigen.

[0023] In some aspects, methods and compositions to improve the anti-tumor efficacy of T-cells (T-lymphocytes) for use in adoptive cellular cancer immunotherapy, comprising suppression of autophagy in said T-cells are provided.

[0024] In one aspect, methods to improve the anti-tumor efficacy of chimeric antigen receptor (CAR) T-cells (CAR-T) for therapy of cancer by suppressing autophagy in said CAR-T-cells are provided.

[0025] In one aspect, the novel use of gene-editing methods (including but not limited to CRISPR-Cas9) to insert CAR nucleic-acid sequences at the loci of autophagy (ATG) genes in primary human autologous T-cells to generate 'knock-in' CAR-T-cells deficient in autophagy is provided. These CAR-T-cells are deficient in expression of the ATG gene targeted by the CAR sequences using CRISPR-Cas9 and are believed to have improved anti-tumor efficacy (against tumors specifically targeted by the CAR-T) by virtue of the ablated or suppressed ATG gene.

[0026] In one aspect, the T-cell autophagy (ATG) gene targeted for'knock-in' of the CAR sequences using CRISPR-Cas9 gene editing (or targeted for knock-out or suppression in other aspects) may be any autophagy-related (ATG) gene known by those skilled in the art and may include but are not limited to ATG1, ATG4, ATG5, ATG7, ATG8, ATG13, ATG4, ATG18, ATG16L, and GABARP. In some aspects, the autophagy gene that is suppressed in the lymphocyte is one or more of ULK, ULK2, ULK3, FIP200, Vps34, Beclin-1, p150, UVRAG, ATG1, ATG4, ATG5, ATG7, ATG8, ATG9, ATGIO, ATG12, ATG13, ATG14L, ATG16L, ATG16LI, ATG18, VMPI, GABARAP, or the like.

[0027] In one aspect, the CAR sequences used in the CRISPR-Cas9 knock-in at the chosen ATG gene locus of the T-cell may be directed to (specific for) any desired tumor (or disease/target) antigen. In some aspects, the antigen targeted by the antigen targeting receptor is a tumor-specific antigen including but not limited to CD19, CD20, BCMA, Her2, EGFRvIII, PSMA (prostate specific membrane antigen) and the FR (folate receptor) for example. In some aspects, the tumor-specific antigen is the folate receptor (FR), the a-folate receptor, the p-folate receptor, the y-folate receptor, CD19, CD20, CD133, CD138, CEA, Claudin 18.2, EGFR, EGFRvIII, EphA2, EpCAM, GD2, GPC3,

HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, PSMA, Fra, NKG2DI, BCMA, ILl3Ralpha2, LeY, CD70, B7-H3, ROR1, PSCA, or the like.

[0028] In one aspect, an engineered CAR-T-cell in which CAR sequences specific for the alpha (a) folate receptor (aFR) are 'knocked-in' to the ATG5 gene locus of T-cells using CRISPR-Cas9 gene-editing. This generates a novel engineered CAR-T-cell (termed aFR-CAR-T) with ablated ATG5 gene activity/function that has improved anti tumour activity (relative to ATG wild-type T-cells) for treatment of a-FR-expressing cancers including but not limited to ovarian, breast and lung cancer. Expression of the aFR-CAR following targeted integration into the ATG5locus may be driven by the endogenous gene promoter or using heterologous promoters. Methods for nuclease and donor delivery are known by persons skilled in the art and can include electroporation of nucleic acids or ribonucleoprotein (RNP) complexes or recombinant virus-mediated delivery.

[0029] In one aspect, an engineered CAR-T-cell in which CAR sequences specific for the alpha (a) folate receptor (aFR) are 'knocked-in' to the ATG14 gene locus of T-cells using CRISPR-Cas9 gene-editing. This generates an engineered CAR-T-cell (termed aFR-CAR-T) with ablated ATG14 gene activity/function that has improved anti-tumour activity (relative to ATG wild-type T-cells) for treatment of a-FR-expressing cancers including but not limited to ovarian, breast and lung cancer.

[0030] In one aspect, the aFR-CAR sequences for the knock-in are targeted to intron 2 of the ATG5 locus. In one aspect, the aFR-CAR is introduced by any suitable method (for example, via electroporation or suitable lentiviral vector or retroviral vector), and then ATG5 is knocked out. In some such aspects, ATG5 is knocked out at exon 4 or exon 5 of the ATG5 locus.

[0031] In one aspect, the sgRNA construct design for the CRISPR-Cas9-mediated knock-in to the ATG5 locus has the nucleotide sequence of SEQ ID NOs:1-7 OR 24-25.

[0032] In one aspect, the primary T-cells used to generate CAR-T knock-ins at various ATG gene loci of the invention, may be induced pluripotent stem cells, CD34+, CD4+ or CD8+ T-cells.

[0033] In one aspect, more than one ATG loci/gene (i.e. two or more different ATG genes) may be simultaneously edited for knock-in by a particular CAR sequence/construct for improved anti-tumour efficacy.

[0034] In one aspect, the aFR-CAR sequences for the knock-in are targeted to introns or exons (i.e. exons 1, 2, 3, 4 etc.) other than intron 2 of the ATG5 locus/gene.

[0034a] Without being taken to limit the generality of the foregoing, the present invention particular provides:

1. A lymphocyte comprising an antigen targeting receptor and a suppressed autophagy gene, wherein the antigen targeting receptor comprises a synthetic receptor.

2. A lymphocyte as defined in 1, comprising a nucleic acid encoding the antigen targeting receptor inserted within a locus of the autophagy gene to disrupt expression of the autophagy gene.

3. A lymphocyte as defined in the 2, wherein the nucleic acid encoding the antigen targeting receptor is inserted within the locus of the autophagy gene so that expression of the antigen targeting receptor is regulated by an endogenous promoter of the autophagy gene.

4. A lymphocyte as defined in any one of 1 to 3, wherein the nucleic acid encoding the antigen targeting receptor is inserted within an exon of the autophagy gene.

5. A lymphocyte as defined in any one of 1 to 3, wherein the nucleic acid encoding the antigen targeting receptor is inserted within an intron of the autophagy gene, optionally wherein the autophagy gene is ATG5 and the nucleic acid encoding the antigen targeting receptor is inserted within intron 2 of the ATG5 gene, and further optionally where the nucleic acid used to insert the antigen targeting receptor has the sequence of one of SEQ ID NO:8 or SEQ ID NO:9.

6. A lymphocyte as defined in 1, wherein the autophagy gene is at a first locus of a genome of the lymphocyte and the lymphocyte comprises a nucleic acid encoding the antigen targeting receptor at a second locus of the genome, the first locus being different from the second locus.

7A (followed by 7B)

7. A lymphocyte as defined in 6, wherein expression of the antigen targeting receptor is regulated by a heterologous promoter.

8. A lymphocyte as defined in any one of 1 to 7, wherein the autophagy gene is ablated, or wherein the autophagy gene is suppressed by RNAi.

9. A method of conducting immunotherapy comprising administering a lymphocyte as defined in any one of 1 to 8 to a subject.

10. A method of conducting immunotherapy using lymphocytes, the method comprising administering to a subject lymphocytes that have been modified to suppress an autophagy gene, optionally wherein the subject is a mammal, and optionally wherein the subject is a human.

11. A method of conducting immunotherapy using lymphocytes, the method comprising administering to a subject lymphocytes that have been modified to express an antigen targeting receptor and to suppress an autophagy gene.

12. A method of treating cancer, the method comprising a step of conducting immunotherapy as defined in any one of 9-11, wherein optionally the cancer is B cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphoid malignancy, liver, pancreatic, brain, breast, ovarian, colorectal, acute myeloid leukemia (AML), multiple myeloma, lung, gastric, glioma, EGFR-positive solid tumor, glioblastoma, glioblastoma multiforme, stomach, nasopharyngeal, esophageal, prostate, neuroblastoma, hepatocellular, squamous cell lung, MSLN-positive solid tumor, non-small-cell lung (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumor, renal cell, central nervous system, or an ROR1-positive malignancy; and optionally wherein the cancer is ovarian cancer, breast cancer, or lung cancer.

13. A method of making a lymphocyte for use in immunotherapy, the method comprising a step of modifying the lymphocyte to suppress an autophagy gene, the method further comprising modifying the lymphocyte to express a synthetic antigen targeting receptor.

7B (followed by 7C)

14. A method as defined in 13, wherein a gene editing method is used to insert a nucleic acid encoding the antigen targeting receptor at a locus of the autophagy gene, optionally wherein the gene editing method is used to insert the nucleic acid encoding the antigen targeting receptor so that expression of the antigen targeting receptor is regulated by an endogenous promoter of the autophagy gene.

15. A method as defined in 13, wherein the step of modifying the lymphocyte to express the antigen targeting receptor comprises inserting a nucleic acid encoding the antigen targeting receptor within a genome of the lymphocyte at a first locus, and wherein the step of modifying the lymphocyte to suppress an autophagy gene comprises knocking out the autophagy gene at a second locus of the genome, the second locus being different from the first locus.

16. A lymphocyte or method as defined in any one of 1 to 15, wherein: the lymphocyte comprises a lymphocyte with activity in killing tumor cells; the lymphocyte comprises a cytotoxic lymphocyte; or the lymphocyte comprises a T-cell, a natural killer (NK) cell, or a B-cell, optionally wherein the T-cell comprises an induced pluripotent stem cell, a CD34+ T-cell, a CD4+ T-cell, a CD8+ T-cell, a Treg cell, a tissue-resident memory T-cell (TRM), or a natural killer T-cell (NKT).

17. A lymphocyte or method as defined in any one of 1 to 16, wherein the autophagy gene is ULKI, ULK2, ULK3, FIP200, Vps34, Beclin-1, p150, UVRAG, ATGI, ATG4, ATG5, ATG7, ATG8, ATG9, ATGIO, ATG12, ATG13, ATG14L, ATG16L, ATG16LI, ATG18, VMPI, or GABARAP; and optionally wherein the autophagy gene is ATG5 or ATG14.

18. A lymphocyte or method as defined in any one of 1 to 17, wherein the antigen targeting receptor comprises a chimeric antigen receptor (CAR) or an engineered T-cell receptor.

19. A lymphocyte or method as defined in any one of 1 to 18, wherein the antigen targeting receptor is specific for a tumor-specific antigen, optionally wherein the tumor-specific antigen comprises the folate receptor (FR), the a-folate receptor, the p-folate receptor, the y-folate receptor, CD19, CD20, CD133, CD138, CEA, Claudin 18.2, EGFR, EGFRvIII, EphA2, EpCAM, GD2, GPC3, 7D (followed by 8)

HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, prostate specific membrane antigen (PSMA), Fra, NKG2DI, BCMA, IL13Ralpha2, LeY, CD70, B7-H3, ROR1, or PSCA.

20. A lymphocyte or method as defined in any one of 1 to 19, wherein the antigen targeting receptor comprises a chimeric antigen receptor (CAR), and wherein the CAR is an a-folate receptor CAR, and wherein optionally the nucleotide construct used to insert the CAR in the lymphocyte has the nucleic acid sequence of either one of SEQ ID NO:8 or SEQ ID NO:9.

[0035] Further aspects of the invention will become apparent from consideration of the ensuing description of preferred embodiments of the invention. A person skilled in the art will realise that other embodiments of the invention are possible and that the details of the invention can be modified in a number of respects, all without departing from the inventive concept. Thus, the following drawings, descriptions and examples are to be regarded as illustrative in nature and not restrictive.

Brief Description of the Drawings

[0036] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0037] FIG. 1 is a schematic diagram showing schematically an example embodiment of a chimeric antigen receptor (CAR).

[0038] FIG. 2 is a schematic diagram of an embodiment in which the DNA encoding the CAR has been inserted at a locus of the genome of the cytotoxic lymphocyte cell at a location other than the locus of the autophagy gene that has been disrupted.

[0039] FIG. 3 is a schematic diagram of an embodiment in which the DNA encoding the CAR has been inserted at the locus of an autophagy gene within the genome of a cytotoxic lymphocyte cell.

[0040] FIG. 4 shows an example embodiment of a method for producing genetically engineered lymphocytes for use in immunotherapy.

[0041] FIG. 5 shows another example embodiment of a method for producing genetically engineered lymphocytes for use in immunotherapy.

[0042] FIG. 6 shows another example embodiment of a method for producing genetically engineered lymphocytes for use in immunotherapy.

[0043] FIG. 7, Panels A-E, show the results of an example demonstrating that autophagy enhances anti-tumor immunity and that this effect is dependent on T-cells.

[0044] FIG. 8, Panels A-K, shows the metabolomics of Atg5 CD8+ T-cells.

[0045] FIG. 9, Panels A and B, show the results of an example demonstrating that Atg5 deficiency leads to increased effector memory CD8+ T-cells.

[0046] FIG. 10, Panels A-E, show the results of an example demonstrating that Atg5-/ CD8+ T-cells have enhanced anti-tumor function.

[0047] FIG. 11, Panels A-J, show the results of experiments demonstrating that Atg5-/- T cells have changes in histone trimethylation and increase in methylation at immune response gene loci.

[0048] FIG. 12, Panels A-1, show a CRISPR-Cas9 strategy and validation of its successful implementation for gene-editing at the ATG5 locus to target the CAR to the ATG5locus with a concomitant functional knock out of ATG5 and targeted integration of a gene trap vector using CRISPR-Cas9.

[0049] FIG. 13 shows the results of experiments demonstrating the delivery of Cas9 RNP targeting ATG5 in primary T-cells.

[0050] FIG. 14 shows the expression of an example aFR-CAR construct on human T-cells.

[0051] FIG. 15, Panels A-E, show the results of experiments demonstrating efficient targeting of ATG5 and AAVS1 in CD34+ hematopoietic stem cells and activated CD8+ T cells.

[0052] FIG. 16 shows the incorporation of the desired aFR-CAR targeting ATG5 into intron 2 of A TG5 in CD8+ T-cells.

[0053] FIG. 17 shows the strategy for incorporating a desired aFR-CAR construct transduced by a lentiviral vector into the T-cell genome followed by electroporation of A TG5 sgRNA into exon 4 or exon 5 of the ATG5 gene to knock out ATG5. Panel A shows the strategy for incorporating the construct into T-cells and knocking out A TG5. Panel B shows the results of TIDE analysis showing the successful deletion at exon 4 of ATG5.

Description

[0054] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense.

[0055] The inventors have now found methods and compositions to improve the anti-tumor efficacy of lymphocytes that have application, for example, in chimeric antigen receptor (CAR) T-cell therapy or T-cell receptor-engineered T-cell therapy. Gene-editing methods for the ablation of autophagy gene expression in lymphocytes including CAR-T-cells have been developed to enhance the therapeutic efficacy of the cells, including against solid tumors. In some embodiments, the gene-editing methods cause the engineered lymphocytes, such as CAR-T-cells, to specifically target the alpha-folate receptor (aFR) in order to improve their effectiveness for treatment of cancers that express the aFR such as ovarian, breast and lung cancer.

[0056] In one aspect, the inventors have designed a novel CRISPR-Cas9 gene-editing strategy to engineer autophagy-deficient lymphocytes, including CAR-T-cells, directed against the ovarian cancer antigen folate receptor alpha (aFR) as an exemplary tumor specific antigen. By using a gene-trap approach to target the aFR chimeric antigen receptor (CAR) as an exemplary antigen targeting receptor into the locus of an exemplary autophagy gene, autophagy-related gene 5 (ATG5), the inventors have disrupted autophagy while concurrently placing the CAR under control of the endogenous A TG5 promoter. Expression of the CAR is therefore upregulated in areas of hypoxia and stress, such as in the solid tumor microenvironment.

[0057] Without being bound by theory, it is believed that placing the antigen targeting receptor, e.g. CAR construct or engineered T-cell receptor (TCR) construct, under control of a promoter, such as the endogenous autophagy gene promoter, that induces expression in response to stress, means that the level of expression of the antigen targeting receptor (e.g. CAR or TCR) will be low in the periphery of the body, but will increase in the tumor microenvironment. This could potentially reduce the toxicity associated with a high dose of cells expressing the antigen targeting receptor, e.g. CAR-T-cells. As an example, ATG5 is constitutively expressed at a low level, but its level of expression increases in response to stress.

[0058] Further without being bound by theory, autophagy deficient mouse T-cells do not generate memory after in vivo infection (25). Without being bound by theory, this may be beneficial for the safety of treatment using the genetically engineered lymphocytes because after the modified cells reach the peak of response, they will die (i.e. because there is no memory formation), thereby clearing all of the genetically engineered lymphocytes from the subject. This provides a measure of safety in administering treatment to patients.

[0059] In one embodiment, the inventors have determined that Atg5 deficient mice have significantly enhanced anti-tumor activity against hormone insensitive prostate and estrogen positive breast tumor cell lines. This anti-tumor response is dependent on T-cells and can be fully replicated with animals defective in other Atg genes (e.g. Atg14, ATG16LI). When T-cells deficient in Atg5 were used in an adoptive T-cell therapy experiments, the inventors observed a therapeutic effect on tumors when compared to wild-type CD8+ T-cells. Thus, the loss of the T-cell intrinsic ATG5 or other autophagy genes has been demonstrated to result in enhanced anti-tumor immune responses.

[0060] Although there are defects of Atg5 loss in other immune subsets including antigen presentation cells, without being bound by theory, the specific loss of autophagy in T-cells may have several benefits for T-cell therapy. Atg5 T-cells from tumor bearing mice have been found to have high rates of glycolysis and low oxidative metabolism. This could be a metabolic advantage as increased glucose metabolism could make tumor infiltrating lymphocytes (TIL) more competitive for glucose. These metabolic changes are associated with Atg5-4T-cells skewing towards effector cells with enhanced antigen specific T-cell responses. Without being bound by theory, the reduced proliferation of Atg5 T-cells may have unintended benefits. Since Atg5 T-cells are highly antigen specific, infusions would require fewer cells to achieve equivalent anti-tumor responses and recipient patients may not need to have lymphodepletion; a procedure that can have significant risk of adverse events. Another feature of Atg5-V T-cells is the metabolic-dependent change in histone trimethylation at specific loci of immune response genes and increase in transcriptional expression of those targets.

[0061] Such benefits provided by the suppression of an autophagy gene can be extended to other lymphocytes and other cells involved in tumor killing. Examples of such cells include NK cells; various types of T-cells including CD34+, CD4+ or CD8+ T-cells, Treg cells, tissue-resident memory T cells (TRM cells), natural killer T (NKT) cells; B-cells, and the like. These cells act through a similar mechanism of recognizing an antigen such as a tumor-specific antigen on the surface of the tumor cell and acting to kill such tumor cell. Without being bound by theory, suppression of autophagy in such cells can enhance effector anti-tumor activity via metabolomics shifts to more glycolysis and greater IFNy secretion. Also in the case of Treg cells, suppression of autophagy could also result in loss of suppressor function against effector T cells.

[0062] In some embodiments, an engineered lymphocyte such as a CAR T-cell having one or more disruptions in an autophagy gene or an otherwise suppressed autophagy gene is provided. In some embodiments, the disruptions in the autophagy gene suppress or ablate expression of the autophagy gene. In some embodiments, the disruption in the autophagy gene is provided using a 'knock-in' genetic engineering strategy. In some embodiments, the disruption in the autophagy gene is provided by using a knock-out genetic engineering strategy. In alternative embodiments, any desired strategy could be used to suppress one or more autophagy genes in the lymphocyte, e.g. a CAR T-cell. Likewise, any suitable genetic engineering strategy could be used to cause the engineered lymphocyte, e.g. CAR T-cell, to express the desired antigen targeting receptor, e.g. a chimeric antigen receptor (CAR) or an engineered T-cell receptor. For example, cells can be transduced with a viral vector (e.g. a lentiviral vector or a retroviral vector) to introduce a nucleotide construct encoding the CAR or engineered T-cell receptor into the cells. In some embodiments, a CRISPR-Cas9 gene editing system (which includes a CRISPR-Cas gene editing system using any suitable Cas protein, e.g. Cas9, Casl2a, or the like) or other similar gene editing technique can be used to disrupt the autophagy gene and/or introduce the antigen targeting receptor construct into the cell for expression. In some embodiments, a combination of different genetic engineering techniques could be used to both disrupt the autophagy gene and introduce the CAR construct into the T-cell for expression. In some embodiments, zinc finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN), Sleeping

Beauty (SB), RNAi, meganucleases, megaTALs, or other gene knockout methods could be used to disrupt or suppress the autophagy gene.

[0063] In some embodiments, the engineered lymphocyte, e.g. CAR T-cell, expresses an antigen targeting receptor, e.g. a chimeric antigen receptor (CAR) or an endogenous or engineered T-cell receptor, that is selective for a tumor-specific antigen. In some embodiments, the tumor-specific antigen is the a-folate receptor (a-FR), which is an antigen that is highly expressed in certain kinds of cancer including ovarian cancer, breast cancer and lung cancer, but which is not expressed at a high level by normal cells. In some embodiments, the tumor-specific antigen is the folate receptor (FR), the p-folate receptor, the y-folate receptor, CD19, CD20, BCMA, Her2, EGFRvIII, or prostate specific membrane antigen (PSMA). In some embodiments, the tumor-specific antigen is the folate receptor (FR), the a-folate receptor, the p-folate receptor, the y-folate receptor, CD19, CD20, CD133, CD138, CEA, Claudin 18.2, EGFR, EGFRvIII, EphA2, EpCAM, GD2, GPC3, HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, PSMA, Fra, NKG2DI, BCMA, IL13Ralpha2, LeY, CD70, B7-H3, ROR1, PSCA, or the like. In alternative embodiments, the tumor-specific antigen targeted by the antigen targeting receptor can be any desired tumor-specific antigen. A non-exhaustive list of exemplary tumor-specific antigens currently being evaluated for treatment of certain cancers is given in Table 1. In some embodiments, the cancer is B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphoid malignancy, liver, pancreatic, brain, breast, ovarian, colorectal, acute myeloid leukemia (AML), multiple myeloma, lung, gastric, glioma, EGFR-positive solid tumor, glioblastoma, glioblastoma multiforme, stomach, nasopharyngeal, esophageal, prostate, neuroblastoma, hepatocellular, squamous cell lung,MSLN-positive solid tumor, non-small-cell lung (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumor, renal cell, central nervous system, or an ROR1-positive malignancy.

Table 1. Exemplary tumor-specific antigens that can be targeted with antigen targeting receptor constructs such as a CAR.

Target Cancer(s) Antigen B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic CD19 leukemia (CLL), and B-cell lymphoma

CD20 lymphoid malignancies liver, pancreatic, brain, breast, ovarian, colorectal, acute myeloid CD133 leukemia (AML) CD138 multiple myeloma CEA lung, colorectal, gastric, breast, pancreatic Claudin 18.2 gastric, pancreatic EGFR EGFR-positive solid tumors, glioma, colorectal EGFRvIII glioblastoma multiforme EphA2 malignant glioma liver, stomach, nasopharynx, colon, esophageal, pancreatic, prostate, EpCAM gastric, hepatic, recurrent breast GD2 neuroblastoma GPC3 hepatocellular, liver, squamous cell lung

HER2 breast, ovarian, lung, gastric, colorectal, glioma, pancreatic MSLN pancreatic, MSLN-positive solid tumors MG7 liver glioma, colorectal, gastric, hepatocellular, non-small-cell lung MUC1 carcinoma (NSCLC), pancreatic, breast, ovarian NY-ESO-1 NSCLC LMP1 nasopharyngeal PSMA prostate Fra ovarian NKG2DL colorectal, triple-negative breast cancer (TNBC), sarcoma BCMA multiple myeloma IL13Ralpha2 glioblastoma LeY advanced solid tumors CD70 pancreatic, renal cell, breast B7-H3 central nervous system (CNS), glioma ROR1 ROR1+ malignancies PSCA prostate

[0064] In some embodiments, the antigen targeting receptor, e.g. CAR construct or endogenous or engineered T-cell receptor (TCR) construct, is inserted at the locus of the autophagy gene that is to be suppressed using a knock-in strategy. This places the antigen targeting receptor, e.g. CAR construct or TCR construct, under control of the endogenous promoter of the autophagy gene. In some embodiments, the antigen targeting receptor, e.g. CAR construct or TCR construct, is inserted in the lymphocyte's (e.g. T-cell) genome at a locus other than the autophagy gene that is to be suppressed. In some such embodiments, expression of the antigen targeting receptor, e.g. CAR construct or TCR construct, is controlled by a heterologous promoter. In some embodiments, the antigen targeting receptor, e.g. CAR construct or TCR construct, is inserted together with a desired exogenous promoter to control expression of the antigen targeting receptor, e.g. CAR or TCR, by the lymphocyte (e.g. T-cell) under the control of an exogenous promoter. In some embodiments, the antigen targeting receptor (e.g. CAR or TCR construct) is placed under control of a promoter that increases expression of the antigen targeting receptor in response to stress. In some embodiments, the promoter that increases expression of the antigen targeting receptor in response to stress is a promoter of an autophagy gene.

[0065] In some embodiments, rather than expressing a CAR, the lymphocyte is engineered to express an endogenous or engineered T-cell receptor as the antigen targeting receptor. TCR-engineered T-cells are currently being developed for use in various types of immunotherapy, including the treatment of solid tumors, and can be used in a manner similar to CAR-T cells to specifically target and kill cells expressing a particular antigen, e.g. a tumor-specific antigen.

[0066] In some embodiments, the autophagy gene (ATG) that is suppressed in the lymphocyte is any gene that is now known or is later discovered to be essential for autophagy. In some embodiments, the autophagy gene is one or more of ATG1, ATG4, ATG5, ATG7, ATG8, ATG13, ATG18, ATG16LI, and GABARAP. In some embodiments, the autophagy gene is one or more of ULK, ULK2, ULK3, FIP200, Vps34, Beclin-1, p150, UVRAG, ATG1, ATG4, ATG5, ATG7, ATG8, ATG9, ATGIO, ATG12, ATG13, ATG14L, ATG16L, ATG16LI, ATG18, VMP, GABARAP, or the like.

[0067] In some embodiments, the CAR construct has a targeting moiety, a transmembrane domain and a CD3( intracellular domain. In some embodiments, the targeting moiety is an antigen-binding fragment of an antibody. In some embodiments, the targeting moiety is an ScFV of an antibody. In alternative embodiments, any CAR construct now known or later developed could be used.

[0068] In alternative embodiments, an engineered T-cell receptor is used as the antigen targeting receptor. An example of an engineered T-cell receptor has an a chain and a chain, each containing a variable domain (v) and a constant domain (c), as well as a transmembrane domain and 6 CD3 chains for T-cell activation. In alternative embodiments, any T-cell receptor construct now known or later developed could be used.

[0069] An example embodiment of a CAR construct 30 for use in some embodiments is shown in FIG. 1. The CAR construct has a targeting moiety 32, e.g. a suitable ScFV that targets a desired tumor-specific antigen, a transmembrane domain 34, and a CD3( intracellular domain 38. In some embodiments, CAR construct 30 can have other domains such as a suitable co-stimulatory domain 36 (e.g. CD27, CD28, 4-1BB, ICOS, OX40, MYD88, IL1R1, CD70, or the like), or other domains intended to enhance the characteristics of the CAR construct.

[0070] FIG. 2 shows schematically an example embodiment of an engineered lymphocyte 50. Engineered lymphocyte 50 has been genetically modified to suppress one or more genes essential for autophagy at a locus 52 of the genomic DNA of the lymphocyte. The gene essential for autophagy is under control of an endogenous promoter 56. Engineered lymphocyte 50 has also been genetically modified to express a desired antigen targeting receptor construct at a locus 54 of the genomic DNA of the lymphocyte which is different from locus 52. The expression of the antigen targeting receptor construct from locus 54 is under control of a promoter 58, which can be an endogenous promoter in some embodiments or a heterologous promoter in some embodiments.

[0071] FIG. 3 shows schematically an example embodiment of an engineered lymphocyte 70. Engineered lymphocyte 70 has been genetically modified to both suppress one or more genes essential for autophagy and express a desired antigen targeting receptor construct at a locus 72 of the genomic DNA of the lymphocyte. For example, a knock-in strategy can be employed to both disrupt the gene essential for autophagy and insert the desired antigen targeting receptor construct at locus 72 of the genomic DNA of the lymphocyte. In this embodiment, expression of the desired antigen targeting receptor construct from locus 72 is under control of the endogenous promoter 76 of the gene essential for autophagy.

[0072] Engineered lymphocytes 50, 70, or modified lymphocytes, including engineered T cells, according to any other embodiment, can be made via any suitable genetic engineering technique now known or later made available to one skilled in the art. In one example embodiment shown in FIG. 4, a method 100 of creating modified lymphocytes such as T-cells using a gene editing strategy to insert the desired antigen targeting receptor construct at the locus of the autophagy gene to be disrupted is shown. At step 102, the desired lymphocytes such as T-cells are obtained from a source of cells. The source of the cells may be any suitable source, for example, the subject to whom the immunotherapeutic lymphocytes are to be administered, a healthy donor, a pluripotent stem cell line, or the like. In some embodiments, the lymphocytes are autologous cells, i.e. the lymphocytes are acquired from the subject to be treated. In some embodiments, the lymphocytes are allogenic, i.e. obtained from source of cells other than the subject to be treated, such as a healthy donor or a cell line such as induced pluripotent stem cells. Any source of lymphocytes now known or later discovered can be used in certain embodiments.

[0073] At step 104, the lymphocytes are genetically engineered to insert DNA encoding the antigen targeting receptor construct in the genomic DNA of the lymphocytes and suppress the function of at least one autophagy gene. In some embodiments, step 104 is carried out using a CRISPR-Cas gene editing strategy, by devising suitable sgRNA to insert the antigen targeting receptor construct at an appropriate location within an autophagy gene so that the antigen targeting receptor construct can be inserted and the autophagy gene simultaneously suppressed using a knock-in strategy. The sgRNA, DNA encoding the antigen targeting receptor construct, and a Cas protein such as Cas9 are delivered to the lymphocyte in any suitable manner, for example via electroporation or chemical transfection techniques, or other suitable techniques as appropriate, for example using a delivery system based on a lentivirus, adenovirus, or adeno-associated virus.

[0074] At step 106, the genetically engineered lymphocytes in which the antigen targeting receptor construct has been successfully inserted at the correct locus are expanded. At step 108, the engineered lymphocytes are introduced into the subject as an immunotherapeutic.

[0075] FIG. 5 shows an alternative embodiment of a method 150 of creating modified lymphocytes such as T-cells for immunotherapy. At step 152, lymphocytes are obtained from a source of cells in a similar manner as described for step 102. At step 154, the lymphocytes, e.g. T-cells, are genetically engineered in any suitable manner to suppress one or more autophagy genes. Examples of techniques that can be used to suppress the autophagy gene include ablation via gene knock out techniques using CRISPR-Cas, TALEN, ZFN, SB, meganucleases, megaTALs, or suppression via gene knockdown via RNAi, or other suitable techniques.

[0076] At step 156, in some embodiments, the genetically engineered lymphocytes in which the autophagy gene has been suppressed are expanded. At step 158, the lymphocytes are introduced into the subject as an immunotherapeutic.

[0077] FIG. 6 shows an alternative embodiment of a method 170 of creating modified lymphocytes such as T-cells for immunotherapy. Steps 172 and 174 are carried out in like manner to steps 152 and 154 of method 150 to produce lymphocytes, e.g. T-cells, that are genetically engineered to suppress one or more autophagy genes. At step 176, the lymphocytes, e.g. T-cells, are further genetically engineered in any suitable manner to express a desired antigen targeting receptor, such as a CAR construct or engineered T-cell receptor construct. Examples of techniques that may be used to engineer the lymphocytes, e.g. T-cells, to express a desired antigen targeting receptor such as a CAR construct or engineered T-cell receptor include CRISPR-Cas editing systems or transduction with a suitable lentiviral or retroviral vector. At step 178, the lymphocytes, e.g. T-cells, that have been genetically engineered to have both a suppressed autophagy gene and to express the desired antigen targeting receptor, e.g. CAR construct or engineered T-cell receptor, are expanded. At step 180, the expanded cells are introduced into the subject as an immunotherapeutic. In alternative embodiments, the order in which steps 174 and 176 are carried out can be reversed, that is the lymphocytes, e.g. T-cells, can be first engineered to express the desired antigen targeting receptor, e.g. CAR construct or engineered T-cell receptor, and then engineered to suppress one or more autophagy genes.

[0078] In some embodiments, the genetically engineered lymphocytes, e.g. T-cells, are used as an immunotherapeutic to treat cancer. In some embodiments, the cancer is ovarian cancer, breast cancer, or lung cancer. In some embodiments, the cancer is B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphoid malignancy, liver, pancreatic, brain, breast, ovarian, colorectal, acute myeloid leukemia (AML), multiple myeloma, lung, gastric, glioma, EGFR-positive solid tumor, glioblastoma, glioblastoma multiforme, stomach, nasopharyngeal, esophageal, prostate, neuroblastoma, hepatocellular, squamous cell lung,MSLN-positive solid tumor, non-small-cell lung (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumor, renal cell, central nervous system, or an ROR1-positive malignancy. The genetically engineered lymphocytes, e.g. T-cells, can be administered to the subject in any suitable manner, for example via intravenous diffusion.

[0079] In some embodiments in which a CRISPR-Cas strategy is used to introduce the antigen targeting receptor construct into the lymphocyte for expression, the CRISPR-Cas strategy is also used to simultaneously suppress expression of an autophagy gene. Persons skilled in the art can develop appropriate single guide RNAs (sgRNAs) to effect the insertion of the antigen targeting receptor, e.g. CAR, at a desired location in the genome of the lymphocyte. In some embodiments, the CRISPR-Cas strategy is used to insert the antigen targeting receptor, e.g. CAR, within the locus of the ATG5 gene in the genome of the lymphocyte. In some embodiments, the CRISPR-Cas strategy is used to insert the antigen targeting receptor, e.g. CAR, within intron 2 of the ATG5 gene. In some embodiments, the CRISPR-Cas strategy is used to disrupt the ATG5 gene at exon 4 or exon 5 of the ATG5 gene while the antigen targeting receptor is inserted at a different locus within the genome, for example using transduction with an appropriate lentiviral, retroviral or adeno-associated viral vector. In some embodiments, the CRISPR-Cas strategy is used to insert DNA encoding an engineered T-cell receptor rather than DNA encoding a CAR.

[0080] In one example embodiment, the sgRNA used to target intron 2 of ATG5 has the sequence of one of SEQ ID NOs:1-7 listed in Table 2. In one example embodiment, the sgRNA used to target exon 4 of ATG5 has the sequence of SEQ ID NO:24. In one example embodiment, the sgRNA used to target exon 5 of ATG5 has the sequence of SEQ ID NO:25.

[0081] In one example embodiment, the nucleotide construct used to insert the CAR into the T-cells has one of SEQ ID NO:8 or SEQ ID NO:9.

Examples

[0082] Certain embodiments are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature. While the following examples demonstrate the suppression of an autophagy gene and expression of a CAR in T-cells, the techniques described below are equally applicable to conduct the genetic engineering of other types of lymphocytes to suppress expression of an autophagy gene and express a desired CAR or engineered T-cell receptor.

[0083] The inventors designed and tested several single guide RNAs (sgRNAs) targeted to the locus of autophagy-related gene 5 (ATG5), and quantified indel formation by sequencing and mismatch cleavage assay. The inventors optimized conditions for homology-directed repair (HDR) in K562 cells using a fluorescent reporter construct. Out out polymerase chain reaction (PCR) and Western blot were used to evaluate construct integration and autophagy activity. To identify transfection parameters that would yield optimal editing and expansion in human T-cells, the inventors then performed a series of electroporations with sgRNA and recombinant Cas9 protein. The inventors used these electroporation parameters in combination with adeno-associated virus (AAV) vectors to target the aFR chimeric receptor into the locus of ATG5 in healthy donor T-cells.

[0084] The inventors observed loss of functional autophagy in clones with donor integration. Furthermore, the inventors confirmed that these results were due to on-target editing by delivering A TG5 cDNA to an intergenic locus and restoring autophagy function. After screening multiple transfection parameters (cell density, RNP concentration, electroporation pulse code, and addition of an anionic polymer) the inventors identified a set of conditions for RNP delivery that yielded upwards of 80% indel formation in healthy donor T-cells. Using these parameters in conjunction with an AAV vector encoding the aFR CAR, the inventors successfully generated 24.1% ATG5 aFR-CAR-T-cells by CRISPR-mediated homology directed repair.

[0085] FIG. 7 shows the results of experiments demonstrating that deletion of autophagy enhances anti-tumor immunity and is dependent on T-cells. The inventors made an inducible Cre-ERT2 mouse to delete Atg5 in all tissues upon tamoxifen injection. 4 days post tamoxifen treatment, e0771 breast (Panel A) or TRAMP-C2 prostate (Panel B) tumor cell lines were injected subcutaneously and tumor volume measured over time. n=12-13 mice per experimental group.

[0086] Panel C shows using a different autophagy gene, Atg14, that Atg14-dependent enhancement of anti-tumor immunity is cell-intrinsic to the T-cells. The inventors performed bone-marrow chimera experiments where wild type (WT) mice were reconstituted with Atg5 or Atg5/ bone marrow (BM). Atg5 BMC mice show enhanced tumor rejection. n=10 mice per experimental group. Similar experiments were performed using Atgl4 or Atgl4+/ BM (data not shown).

[0087] Panel D shows that antibody depletion of CD8+ T-cells (achieved using an anti-CD8 antibody) results in loss of tumor control in Atg5-/- mice. n=4-7 mice per experimental group. ****p<0.0001, two-way ANOVA. Error bars indicate +/- SEM. Panel E shows the adoptive transfer of naive CD8+ Atg5 deficient (AA) or Atg5 wildtype (Aa) T-cells into EG7 tumor bearing mice. Tumors were implanted and donor T-cells transferred on Day 11 (ACT). **** p <0.0001, Student's t test. Error bars indicated +/- SD.

[0088] FIG. 8 shows the results of experiments evaluating the metabolomics of Atg5 CD8+ T-cells and showing that Atg5 CD8+ T-cells are more glycolytic. *p<0.01, **p<0.01, ***p<0.001, ****p<0.001. Panel A shows the relative lactate levels identified by

metabolomics. The error bars indicate +/- SD. Panel B shows oxygen consumption rate (OCR) results for CD8+ T-cells that were isolated from spleens of e0771 tumour bearing mice and were subjected to Seahorse Bioanalyzer in the presence or absence of oligomycin, FCCP and Antimycin/Rotenone (Ant/Rot). Data was normalized to protein concentration. Error bars indicate +/- SEM. OCR (Panel C), extracellular acidification rate (ECAR) (Panel D) and OCR:ECAR (Panel E) ratio of Atg5+/ and Atg5 CD8+ T-cells was examined at basal levels. Atg5 T-cells exhibit an increased oxygen consumption rate and increased extracellular acidification rate.

[0089] Panel F on the left shows a representative flow cytometry plot showing fluorescent glucose analog 2-NBDG uptake in splenic Atg5+/ (lower trace) and Atg5 (upper trace) CD8+ T-cells from e0771 tumour bearing mice. Graph on the right half of Panel F represents the mean fluorescence intensity (MFI) of 2-NBDG +/- SEM, with Atg5 exhibiting considerably higher fluorescence and therefore higher glucose uptake.

[0090] Panel G shows quantitative RT-PCR of Hxk2 expression in Atg5+/ and Atg5 CD8+ T-cells. Results are relative to Actb. Data are expressed as average +/- SEM. Panel H shows spare respiratory capacity, indicated by baseline OCR subtracted from maximal OCR, and Panel I shows mitochondrial mass as measured by MitoTracker Green, as determined in Atg5+/ and Atg5 CD8+ T-cells isolated from e0771 tumour bearing mice. Data are expressed as average +/- SEM. n=4-5 mice per group.

[0091] Panel J shows T metabolomics analysis for cells from tumor bearing Atg5 (n=5) or Atg5'/- (n=4) mice that were harvested on day 14 post-tumor implantation and (10,000 cells). Panel K shows a volcano plot analysis which revealed a significant increase in glycolysis (e.g. lactate) with concomitant reductions in oxidative metabolism (e.g. glutamate, aspartate).

[0092] FIG. 9 shows the results of experiments demonstrating that Atg5 deficiency leads to increased effector memory CD8+ T-cells. Panel A shows representative flow cytometry plots showing naive (CD62LhiCD441o), central memory (CD62LhiCD44hi) and effector memory (CD62LoCD44hi) CD8+ T-cells isolated from blood (left side), spleen (centre) and tumours (right side) of e0771 tumour bearing mice. Panel B shows the percentages of CD62LloCD44hi effector memory CD8+ T-cells in blood, spleen and tumours from e0771 tumour bearing mice. * p<0.05, ** p<0.01.

[0093] With reference to FIG. 10, the inventors have carried out experiments demonstrating that Atg5-/- CD8+ T-cells have enhanced anti-tumor function. The left two images of Panel A show representative flow cytometry plots showing IFNy and TNFa expression following PMA/ionomycin stimulation of Atg5+/ and Atg5 CD8+ T-cells from Tramp-C2 tumour bearing mice. The graphs shown to the right of Panel A represent the percentage of IFNy+TNFa+ CD8+ T-cells in e0771 and Tramp-C2 tumour bearing mice +/- SEM, with the percentage being higher for Atg5 cells. Panel B shows serum from e0771 or Tramp-C2 tumour bearing mice as analyzed by ELISA. Graphs represent the average +/- SEM. Levels of IFNy were higher for Atg5 mice. Panel C shows splenocytes harvested from Atg5'/- and Atg5-/- e0771 tumour bearing mice which were were stimulated with e0771 cells, and IFNy ELISPOT assays were performed. Data are expressed as average +/- SEM. In another experiment, splenocytes harvested from Tramp-C2 tumour bearing mice were stimulated with Tramp-C2 cells (Panel D) or Spas-1 peptide (Panel E), and IFNy ELISPOT assays were performed. Data are expressed as average +/- SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Increased TNFa and IFNy are observed in Atg5 T-cells.

[0094] With reference to FIG. 11, the inventors conducted experiments to show that Atg5- T-cells have changes in histone trimethylation and increase in methylation at immune response gene loci. Panel A shows pathway and gene ontology analysis of H3K4me3 marked promoters unique to Atg5 versus Atg5 1 CD8+ T-cells are strongly enriched in genes related to T-cell activation and adaptive immunity (Benjamini q value <10e-12). Panel B shows differential normalized tagged density of H3K27me3 and H3K4m3 in a 1 and Atg5' subset of immune response genes. Panels C and D show ChIP-Seq on Atg5+ CD8+ T-cells for H3K4me3 for Ifng. Panel C shows normalized tag density ofIfng in knockout or control CD8+ T-cells. Panel D shows quantitative RT-PCR ofIfng expression in Atg14' and Atg14' CD8+ T-cells. Results are relative to Actb. Data are expressed as average +/- SEM of a triplicate experiment with at least 2 mice per group. Panels E-G show ChlP-Seq on Atg5'1 and Atg5'CD8+ T-cells for H3K4me3 for Glut1. Panel E shows normalized tag density of Glut1 in knockout or control CD8+ T-cells. Panel F shows quantitative RT-PCR of Glut1 expression in Atg5*'and Atg5'4CD8+ T-cells or (Panel G) Atg14' and Atg14' CD8+ T-cells. Results are relative to Actb. Data are expressed as average +/- SEM of a triplicate experiment with at least 2 mice per group. Panels H-J show ChlP-Seq on Atg5*' and Atg5V CD8+ T-cells for H3K4me3 for Hk2. Panel H shows normalized tag density of Hk2 in knockout or control CD8+ T-cells. Panel I shows quantitative RT-PCR of Hk2 expression in Atg5'1 and Atg5'CD8+ T-cells or (Panel J) Atg14' and Atg14' CD8+ T-cells. Results are relative to Actb. Data are expressed as average +/- SEM of a triplicate experiment with at least 2-3 mice per group. *p<0.01, **p<0.01, ****p<0.001, n.s. not significant.

[0095] With reference to FIG. 12, a CRISPR-Cas9 strategy and validation for gene-editing at the ATG5locus was developed for the targeting of the CAR to the ATG5 locus. Concomitant functional knock out of ATG5 and targeted integration of a gene trap vector using CRISPR-Cas9 was confirmed. Panel A shows a schematic of the ATG5locus and the regions screened to identify functional SpCas9-sgRNAs. Exons E2 and E3 of the A TG5 gene are shown as open boxes. Genomic structure and target regions within intron 2 (12) of ATG5 are illustrated schematically. A, B, and C denote non-repetitive target DNA sequences used to design sgRNAs using the online CRISPOR tool. Intron 2 was selected as a preferential target region since all reported mRNA isoforms of ATG5 include this region (i.e. this ensures that all potential transcripts are captured).

[0096] Panel B of FIG. 12 shows the sgRNA sequences identified by CRISPOR targeting different regions of intron 2 to arrive at the sgRNA sequences shown in Table 2.

Table 2. Example sgRNA sequences for ATG5 ablation via insertion in intron 2.

Locus Name DNA Sequence (5'-3') SEQ ID NO

12A Target 719 GCTACGGAAAGTCAGATTAC SEQ ID NO:1

12A Target 696 GTAATCTGACTTTCCGTAGC SEQ ID NO:2

12A Target 824 GCACCGAGTAGTACCACTTG SEQ ID NO:3

12A Target 950 AAGTTCGGCAATCTTGTTAC SEQ ID NO:4

12B Target 905 CGGATCGCTGCCTAATGTTA SEQ ID NO:5

12B Target 945 CCGTTTATGTATCCTTAGTC SEQ ID NO:6

12C Target 710 GTCACGTTCTCCTACCTAGT SEQ ID NO:7

[0097] Panel C of FIG. 12 shows the identification of active sgRNAs using the surveyor nuclease assay. In this assay, a mismatch sensitive nuclease is used to determine the frequency of the small insertions and deletions (indels) indicative of nuclease activity. As an example, sgRNA 824 appears to be the most active nuclease for section 12A of intron 2 and was selected for further experiments.

[0098] Panel D of FIG. 12 shows a schematic of the ATG5 locus following CAR addition. The first and second schematics show the genomic structure of the CAR-T integration site and donor template. Shown are the locations of the splice acceptor site (SA), 2A self cleaving peptide sequence (2A), polyadenylation sequence (pA), and homology arms left and right (HA-L, HA-R). The third schematic shows the integrated CAR construct at the A TG5 locus.

[0099] Panel E of FIG. 12 shows out-out PCR showing integration of a test SA-2A mScarlet-pA reporter sequence within intron 2 of ATG5. Complete integration at all alleles is observed for K562 clones 5,6,7,8,10. Panel F is a Western blot showing loss of ATG5 in K562 clones correlates with complete integration of the mScarlet construct (i.e. loss of ATG5 is observed for clones 5,6,7,8,10. Panel G is an immunofluoresence image of K562 cells showing nuclear staining (left) and mScarlet expression (right), showing good expression of the mScarlet reporter construct. Panel H is a Western blot showing response to treatment with hydroxychloroquine and/or rapamycin. Loss of LC3-II indicating inhibited autophagic flux is observed in K562 clones 5,6,7 (clones 8 and 10 were not tested). Panel I is a Western blot showing that for clones for which an ATG5 cDNA was targeted to an intergenic locus (AAVS1) to restore expression of ATG5 (clones 5.1, 5.3 and 5.6), autophagy was restored as confirmed by the return of LC3-II.

[0100] FIG. 13 shows the results of experiments demonstrating delivery of Cas9 RNP targeting ATG5 in primary T-cells. Purified CD3 cells stimulated with CD3/CD28 beads were electoporated with recombinant SpCas9 complexed with a chemically modified sgRNA (824) using the amaxa 4D Nucleofector. Two different pulses were tested. Genomic DNA was extracted 3 days post transfection and the Surveyor assay was performed to determine the frequency of indels.

[0101] FIG. 14 shows the expression of an exemplary aFR-CAR (CD3zBB) with a GFP reporter from Human CD8+ T-cells transduced with lentiviruses encoding the construct. The left panel is empty vector. The right panel is the aFR-CAR construct showing expression of the desired protein on human T-cells.

[0102] The sequences of the CAR constructs used to insert the aFR-CAR at the ATG5 locus are given as SEQ ID NOs:8 and 9The sequences of the CAR constructs used to insert the aFR-CAR within the AAVS1locus as a control are given as SEQ ID NOs:10 and 11. SEQ ID NO:8 encodes a CAR construct having an anti-folate receptor ScFv, a transmembrane domain, a CD27 co-stimulatory domain, and a CD3( intracellular domain. SEQ ID NO:9 encodes a CAR construct having an anti-folate receptor ScFv, a transmembrane domain, a CD28 co-stimulatory domain, and a CD3( intracellular domain. SEQ ID NO:10 encodes a CAR construct having an anti-folate receptor ScFv, a transmembrane domain, a CD27 co-stimulatory domain, and a CD3( intracellular domain. SEQ ID NO:11 encodes a CAR construction having an anti-folate receptor ScFv, a transmembrane domain, a CD28 co-stimulatory domain, and a CD3( intracellular domain.

[0103] FIG. 15 shows the efficient targeting of ATG5 and AAVS1 in CD34+ hematopoietic stem cells and activated CD8+ T-cells (no donor CAR construct was present in these experiments). Panel A is a histogram showing editing efficiency at ATG5 and AAVS1 in cord blood derived CD34+ HSCs after electroporation with Cas9 protein and 100-300 pmol sgRNA as indicated. Genomic DNA from each target region was amplified and sequenced, and the sequence traces analyzed using the decomposition algorithm TIDE. Panels B and C are flow cytometry dot plots showing 47.5% viability (ATG5) and 31.6% viability (AAVSI) in CD8+ T-cells three days after electroporation with a ribonucleoprotein (RNP) complex consisting of Cas9 protein and sgRNA. Panels D and E are histograms showing 54.1% overall editing efficiency at ATG5 and 53.9% overall editing efficiency at AAVS1 in CD8+ T cells as determined by TIDE analysis.

[0104] FIG. 16 shows the validation of the incorporation of the CAR construct into intron 2 of ATG5 in healthy donor T-cells. CD8+ T-cells were isolated from healthy donor PBMCs by magnetic bead separation, stimulated with TransAct (Miltenyi), and cultured in 100U/ml IL-2. On day 4 post-activation, the cells were electroporated with an RNP consisting of 1OOpmol sgRNA targeting ATG5 and 61pmol Cas9 protein. 15 minutes post-electroporation, C4-CD28-CD3z AAV (having SEQ ID NO:9) was given at an MOI of 2e5. The cells were expanded for 13 days and then stained for flow cytometry on a Cytek Aurora. The results show that 24.1% of the cells were the desired ATG5-/- aFR-CAR-T-cells.

[0105] FIG. 17 shows the strategy for incorporating a desired aFR-CAR transduced by a lentiviral vector into the T-cell genome followed by electroporation of sgRNA targeting ATG5 into exon 4 or exon 5 of the ATG5 gene using the sgRNA sequences shown in Table 3. Panel A shows the strategy for incorporating the construct into T-cells and ablating A TG5 at exon 4 (Target 56) or exon 5 (Target 150). Panel B shows the results of TIDE analysis showing the successful deletion at exon 4 of ATG5 using an sgRNA having the sequence of SEQ ID NO:24 (Target 56).

Table 3. Example sgRNA sequences for ATG5 ablation via insertion in exon 4 or exon 5.

Locus Name DNA Sequence (5'-3') SEQ ID NO

E4 Target 56 CATCAAGTTCAGCTCTTCCT SEQ ID NO:24

E5 Target 150 GATCACAAGCAACTCTGGAT SEQ ID NO:25

The foregoing example demonstrates that a desired antigen targeting receptor construct can be integrated into the genome of a lymphocyte such as a T-cell at one locus in the genome, while an autophagy gene such as ATG5 can be knocked out at a different locus using CRISPR-Cas gene editing.

References

[0106] The following references are of interest with respect to the subject matter described herein. Each one of the following references is incorporated by reference herein in its entirety.

1. Geyer MB, Brentjens RJ. 2016. Review: Current clinical applications of chimeric antigen receptor (CAR) modified T-cells. Cytotherapy 18: 1393-409 2. Kershaw MH, et al., 2006. A phase I study on adoptive immunotherapy using gene modified T-cells for ovarian cancer. Clin Cancer Res 12: 6106-15 3. Zhao Z, et al., 2015. Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T-cells. Cancer Cell 28: 415-28 4. Milne K, et al., 2009. Systematic analysis of immune infiltrates in high-grade serous ovarian cancer reveals CD20, FoxP3 and TIA-1 as positive prognostic factors. PLoS One 4: e6412 5. Cheung A, et al., 2016. Targeting folate receptor alpha for cancer treatment. Oncotarget 6. Newick K, et al., 2017. CAR T-cell Therapy for Solid Tumors. Annu Rev Med 68: 139-52 7. Townsend KN, et al., 2013. Markers of T-cell infiltration and function associate with favorable outcome in vascularized high-grade serous ovarian carcinoma. PLoS One 8:e82406 8. Vander Heiden MG, DeBerardinis RJ. 2017. Understanding the Intersections between Metabolism and Cancer Biology. Cell 168: 657-69 9. Chang CH, Pearce EL. 2016. Emerging concepts of T-cell metabolism as a target of immunotherapy. Nat Immunol 17: 364-8 10. Chang CH, et al., 2015. Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression. Cell 162: 1229-41 11. MacPherson S, et al., 2017. STAT3 regulation of citrate synthase is essential during the initiation of lymphocyte cell growth. Cell Rep 19(5):910-918 12. Ma EH, et al., 2017. Serine Is an Essential Metabolite for Effector T-cell Expansion. Cell Metab 25: 345-57

13. Scharping NE, et al., 2016. The Tumor Microenvironment Represses T-cell Mitochondrial Biogenesis to Drive Intratumoral T-cell Metabolic Insufficiency and Dysfunction. Immunity 14. Lum JJ, DeBerardinis RJ, Thompson CB. 2005. Autophagy in metazoans: cell survival in the land of plenty. Nat Rev Mol Cell Biol 6: 439-48 15. Pua HH, He YW. 2009. Autophagy and lymphocyte homeostasis. Curr Top Microbiol Immunol 335: 85-105 16. Schlie K, et al., 2015. Survival of effector CD8+ T-cells during influenza infection is dependent on autophagy. JImmunol194: 4277-86 17. Puleston DJ, et al., 2014. Autophagy is a critical regulator of memory CD8+ T-cell formation. Elife 3 18. Qu X, et al., 2003. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J ClinInvest 112: 1809-20 19. Takamura A, et al., 2011. Autophagy-deficient mice develop multiple liver tumors. Genes Dev 25: 795-800 20. Macintyre AN, et al., 2014. The glucose transporter Glut1 is selectively essential for CD4 T-cell activation and effector function. Cell Metab 20: 61-72 21. Hultquist JF, et al., 2016. A Cas9 Ribonucleoprotein Platform for Functional Genetic Studies of HIV-Host Interactions in Primary Human T-cells. Cell Rep 17: 1438-52 22. Eyquem J, et al., 2017. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543: 113-7. 23. DeVorkin L, et al., 2019. Autophagy regulation of metabolism is required for CD8+ T-cell anti-tumour immunity. Cell Rep 27(2):502-513. 24. Stadtmauer, EA, et al., 2020. CRISPR-engineered T cells in patients with refractory cancer. Science 06 Feb 2020. DOI: 10.1126/science.aba7365. 25. Schlie et al., 2015. Survival of effector CD8+ T-Cells during influenza infection is dependent on autophagy. J.Immunol. 194(9):4277-4286.

[0107] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.

SEQUENCE LISTING SEQUENCE LISTING

<110> Provincial Health Services Authority <110> Provincial Health Services Authority Université Laval Université Laval <120> NOVEL COMPOSITION AND METHODS TO ENHANCE LYMPHOCYTE‐MEDIATED <120> NOVEL COMPOSITION AND METHODS TO ENHANCE LYMPHOCYTE-MEDIATED IMMUNTHERAPY IMMUNTHERAPY

<130> B537 0163/JAM <130> B537 0163/JAM

<150> US 62/804,658 <150> US 62/804,658 <151> 2019‐02‐12 <151> 2019-02-12

<160> 25 <160> 25

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 719 <223> Target 719

<400> 1 <400> 1 gctacggaaa gtcagattac 20 gctacggaaa gtcagattac 20

<210> 2 <210> 2 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 696 <223> Target 696

<400> 2 <400> 2 gtaatctgac tttccgtagc 20 gtaatctgac tttccgtagc 20

<210> 3 <210> 3 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 824 <223> Target 824

<400> 3 <400> 3 gcaccgagta gtaccacttg 20 gcaccgagta gtaccacttg 20

<210> 4 <210> 4

<211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 950 <223> Target 950

<400> 4 <400> 4 aagttcggca atcttgttac 20 aagttcggca atcttgttac 20

<210> 5 <210> 5 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 905 <223> Target 905

<400> 5 <400> 5 cggatcgctg cctaatgtta 20 cggatcgctg cctaatgtta 20

<210> 6 <210> 6 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 945 <223> Target 945

<400> 6 <400> 6 ccgtttatgt atccttagtc 20 ccgtttatgt atccttagtc 20

<210> 7 <210> 7 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 710 <223> Target 710

<400> 7 <400> 7 gtcacgttct cctacctagt 20 gtcacgttct cctacctagt 20

<210> 8 <210> 8 <211> 10041 <211> 10041 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> pAAV_ATG5_C4‐CD27CD3z‐CAR.xdna <223> pAAV_ATG5_C4-CD27CD3z-CAR.xdna

<400> 8 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 00

aggggttcct gcggcctaag cttgagcgga gttccaattg tactgtacag cacatagact 180 00

tgcaggtgtg agttaatgga ttatgtattt tacaaacact tttcaaatgt cttctgattt 240

tggtgtgctt tgtttccctt ttctaagtgc atgtataatc ccagtttatg cctctgtcag 300 00 00

agatgacatc ctgcctgttc actgaatagc ccataatagg caaagatatt ctcaattgtc 360

gttactgttt tttctttttg tgtctcaggt atgcattcta gtgtttacct ggagatgatg 420 00 00

tggaaaagta gaagtgtatt tgtgatgagg aaatgcttct tctgtttcac tttaatgaag 480 00

tctgcccttt gctttccccc tcatctctct gagaggcagt gctgctcctc agcctgctcc 540

agctacggaa agtcagatta ctggaggaag ctacttatta aggagcccat tcgctcactt 600 00

tttgggtata ggactgtttt tttgtttttg ttttcctctg tgcatcctca gcatctggca 660 00

caggctcctc aaaagcttct gacctcttct cttcctccca cagggcctcg agagatctgg 720 bo

cggcggagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc 780

tatggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag 840 bo

gccgggatcc cagctggtgg agtctggggg aggcttggta cagccagggc ggtccctgag 900 00

actctcctgc acaacttctg gattcacttt tggtgattat gctatgatct gggcccgcca 960

ggctccaggg aaggggctgg agtgggtctc atccattagt agtagtagta gttacatata 1020

ctacgcagac tcagtgaagg gccgattcac catctccaga gacaacgcca agaactcact 1080

gtatctgcaa atgaacagcc tgagagccga ggacacggct gtgtattact gtgcgagaga 1140

acgatacgat ttttggagtg gaatggacgt ctggggcaaa gggaccacgg tcaccgtctc 1200

gagtggtgga ggcggttcag gcggaggtgg ctctggcggt agtgcacagt ctgccctgac 1260

tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc accatctcct gcactggaac 1320

cagcagtgat gttgggagtt ataaccttgt ctcctggtac caacagcacc caggcaaagc 1380

ccccaaactc atgatttatg agggcagtaa gcggccctca ggggtttcta atcgcttctc 1440

tggctccaag tctggcaacg cggcctccct gacaatctct gggctccagg ctgaggacga 1500

ggctgattat tactgccagt cctatgacag cagcctgagt gtggtattcg gcggagggac 1560

caagctgacc gtcctaggtg ctagcaccac gacgccagcg ccgcgaccac caacaccggc 1620 caagctgacc gtcctaggtg ctagcaccac gacgccagcg ccgcgaccac caacaccggc 1620 gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg 1680 gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg 1680 gggcgcagtg cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt 1740 gggcgcagtg cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt 1740 ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gccaacgaag 1800 ggccgggact tgtggggtcc ttctcctgtc actggttatc accctttact gccaaccaag 1800 gaaatataga tcaaacaaag gagaaagtcc tgtggagcct gcagagcctt gtcgttacag 1860 gaaatataga tcaaacaaag gagaaagtcc tgtggagcct gcagagcctt gtcgttacag 1860 ctgccccagg gaggaggagg gcagcaccat ccccatccag gaggattacc gaaaaccgga 1920 ctgccccagg gaggaggagg gcagcaccat ccccatccag gaggattacc gaaaaccgga 1920 gcctgcctgc tcccccagag tgaagttcag caggagcgca gacgcccccg cgtaccagca 1980 gcctgcctgc tcccccagag tgaagttcag caggagcgca gacgcccccg cgtaccagca 1980 gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt 2040 gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt 2040 ggacaagaga cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca 2100 ggacaagaga cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca 2100 ggaaggcctg tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg 2160 ggaaggcctg tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg 2160 gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac 2220 gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac 2220 agccaccaag gacacctacg acgcccttca catgcaggcc ctgccccctc gctgataagc 2280 agccaccaag gacacctacg acgcccttca catgcaggcc ctgccccctc gctgataagc 2280 ggccgcctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2340 ggccgcctgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2340 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2400 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2400 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 2460 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggad agcaaggggg 2460 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg ggtcgacgtg 2520 aggattggga agacaatage aggcatgctg gggatgcggt gggctctatg ggtcgacgtg 2520 gtactactcg gtgcatgaat gtttccccag gctcatgttg ggattttgtt tgaaatcatc 2580 gtactactcg gtgcatgaat gtttccccag gctcatgttg ggattttgtt tgaaatcatc 2580 tttatgggtt ttcttactag aaatgatagt tttagagaag ttcggcaatc ttgttactgg 2640 tttatgggtt ttcttactag aaatgatagt tttagagaag ttcggcaatc ttgttactgg 2640 tactcaagct tatatacacg tgagtaatct taactttgga aatatcaacc gttttctgtc 2700 tactcaagct tatatacacg tgagtaatct taactttgga aatatcaacc gttttctgtc 2700 tagaaaaaca tccacagtta aaaaccaagt atggtcatag ttttattttt agaagaattt 2760 tagaaaaaca tccacagtta aaaaccaagt atggtcatag ttttattttt agaagaattt 2760 catttgttag aggtattttt tctgttcttg ttctgtaata taggttagga taccattttt 2820 catttgttag aggtattttt tctgttcttg ttctgtaata taggttagga taccattttt 2820 tttttccaaa caagtttttg ctgttttaat tacaactttt aaccttcatt ggctttgtaa 2880 tttttccaaa caagtttttg ctgttttaat tacaactttt aaccttcatt ggctttgtaa 2880 tagtatataa gagtatgtga gagtataaga gtatatttgg ccaaattgac aacaactggg 2940 tagtatataa gagtatgtga gagtataaga gtatatttgg ccaaattgac aacaactggg 2940 taccctcgag cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 3000 taccctcgag cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 3000 tcgctcactg aggccgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 3060 tcgctcactg aggccgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 3060 gctgcctgca ggggcagctt gaaggaaata ctaaggcaaa ggtactgcaa gtgctcgcaa 3120 gctgcctgca ggggcagctt gaaggaaata ctaaggcaaa ggtactgcaa gtgctcgcaa 3120 cattcgctta tgcggattat tgccgtagtg ccgcgacgcc gggggcaaga tgcagagatt 3180 cattcgctta tgcggattat tgccgtagtg ccgcgacgcc gggggcaaga tgcagagatt 3180 gccatggtac aggccgtgcg gttgatattg ccaaaacaga gctgtggggg agagttgtcg 3240 agaaagagtg cggaagatgc aaaggcgtcg gctattcaag gatgccagca agcgcagcat 3300 00EE atcgcgctgt gacgatgcta atcccaaacc ttacccaacc cacctggtca cgcactgtta 3360 09EE agccgctgta tgacgctctg gtggtgcaat gccacaaaga agagtcaatc gcagacaaca 3420 ttttgaatgc ggtcacacgt tagcagcatg attgccacgg atggcaacat attaacggca 3480 7874 the tgatattgac ttattgaata aaattgggta aatttgactc aacgatgggt taattcgctc 3540 the the the gttgtggtag tgagatgaaa agaggcggcg cttactaccg attccgccta gttggtcact 3600 009E tcgacgtatc gtctggaact ccaaccatcg caggcagaga ggtctgcaaa atgcaatccc 3660 099E gaaacagttc gcaggtaata gttagagcct gcataacggt ttcgggattt tttatatctg 3720 OZLE cacaacaggt aagagcattg agtcgataat cgtgaagagt cggcgagcct ggttagccag 3780 08LE tgctctttcc gttgtgctga attaagcgaa taccggaagc agaaccggat caccaaatgc 3840 the gtacaggcgt catcgccgcc cagcaacagc acaacccaaa ctgagccgta gccactgtct 3900 006E gtcctgaatt cattagtaat agttacgctg cggcctttta cacatgacct tcgtgaaagc 3960 096E the the gggtggcagg aggtcgcgct aacaacctcc tgccgttttg cccgtgcata tcggtcacga 4020 9777780087 acaaatctga ttactaaaca cagtagcctg gatttgttct atcagtaatc gaccttattc 4080 0801 ctaattaaat agagcaaatc cccttattgg gggtaagaca tgaagatgcc agaaaaacat 4140 cheese the gacctgttgg ccgccattct cgcggcaaag gaacaaggca tcggggcaat ccttgcgttt 4200 gcaatggcgt accttcgcgg cagatataat ggcggtgcgt ttacaaaaac agtaatcgac 4260

7 gcaacgatgt gcgccattat cgcctggttc attcgtgacc ttctcgactt cgccggacta 4320

agtagcaatc tcgcttatat aacgagcgtg tttatcggct acatcggtac tgactcgatt 4380 08ED

ggttcgctta tcaaacgctt cgctgctaaa aaagccggag tagaagatgg tagaaatcaa 4440

taatcaacgt aaggcgttcc tcgatatgct ggcgtggtcg gagggaactg ataacggacg 4500 000 tcagaaaacc agaaatcatg gttatgacgt cattgtaggc ggagagctat ttactgatta 4560 00 ctccgatcac cctcgcaaac ttgtcacgct aaacccaaaa ctcaaatcaa caggcgccgg 4620

acgctaccag cttctttccc gttggtggga tgcctaccgc aagcagcttg gcctgaaaga 4680 089/7

cttctctccg aaaagtcagg acgctgtggc attgcagcag attaaggagc gtggcgcttt 4740

the acctatgatt gatcgtggtg atatccgtca ggcaatcgac cgttgcagca atatctgggc 4800 008/7 ttcactgccg ggcgctggtt atggtcagtt cgagcataag gctgacagcc tgattgcaaa 4860 098t attcaaagaa gcgggcggaa cggtcagaga gattgatgta tgagcagagt caccgcgatt 4920 the atctccgctc tggttatctg catcatcgtc tgcctgtcat gggctgttaa tcattaccgt 4980 086/7 gataacgcca ttacctacaa agcccagcgc gacaaaaatg ccagagaact gaagctggcg 5040 aacgcggcaa ttactgacat gcagatgcgt cagcgtgatg ttgctgcgct cgatgcaaaa 5100 00IS tacacgaagg agttagctga tgctaaagct gaaaatgatg ctctgcgtga tgatgttgcc 5160 09TS gctggtcgtc gtcggttgca catcaaagca gtctgtcagt cagtgcgtga agccaccacc 5220 0225 gcctccggcg tggataatgc agcctccccc cgactggcag acaccgctga acgggattat 5280 0825 ttcaccctca gagagaggct gatcactatg caaaaacaac tggaaggaac ccagaagtat 5340 OTES attaatgagc agtgcagata gagttgccca tatcgatggg caactcatgc aattattgtg 5400 agcaatacac acgcgcttcc agcggagtat aaatgcctaa agtaataaaa ccgagcaatc 5460 catttacgaa tgtttgctgg gtttctgttt taacaacatt ttctgcgccg ccacaaattt 5520 7778707778 0255 tggctgcatc gacagttttc ttctgcccaa ttccagaaac gaagaaatga tgggtgatgg 5580 0855 tttcctttgg tgctactgct gccggtttgt tttgaacagt aaacgtctgt tgagcacatc 5640 7877788558 ctgtaataag cagggccagc gcagtagcga gtagcatttt tttcatggtg ttattcccga 5700 00/S tgctttttga agttcgcaga atcgtatgtg tagaaaatta aacaaaccct aaacaatgag 5760 09/9 the ttgaaatttc atattgttaa tatttattaa tgtatgtcag gtgcgatgaa tcgtcattgt 5820 0289 attcccggat taactatgtc cacagccctg acggggaact tctctgcggg agtgtccggg 5880 088S the aataattaaa acgatgcaca cagggtttag cgcgtacacg tattgcatta tgccaacgcc 5940 ccggtgctga cacggaagaa accggacgtt atgatttagc gtggaaagat ttgtgtagtg 6000 0009 the ttctgaatgc tctcagtaaa tagtaatgaa ttatcaaagg tatagtaata tcttttatgt 6060 0909 tcatggatat ttgtaaccca tcggaaaact cctgctttag caagattttc cctgtattgc 6120 tgaaatgtga tttctcttga tttcaaccta tcataggacg tttctataag atgcgtgttt 6180 08t9 cttgagaatt taacatttac aaccttttta agtcctttta ttaacacggt gttatcgttt 6240 777877778 e e tctaacacga tgtgaatatt atctgtggct agatagtaaa tataatgtga gacgttgtga 6300 00E9 cgttttagtt cagaataaaa caattcacag tctaaatctt ttcgcacttg atcgaatatt 6360 0989 tctttaaaaa tggcaacctg agccattggt aaaaccttcc atgtgatacg agggcgcgta 6420 gtttgcatta tcgtttttat cgtttcaatc tggtctgacc tccttgtgtt ttgttgatga 6480 gtttgcatta tcgtttttat cgtttcaatc tggtctgacc tccttgtgtt ttgttgatga 6480 tttatgtcaa atattaggaa tgttttcact taatagtatt ggttgcgtaa caaagtgcgg 6540 tttatgtcaa atattaggaa tgttttcact taatagtatt ggttgcgtaa caaagtgcgg 6540 tcctgctggc attctggagg gaaatacaac cgacagatgt atgtaaggcc aacgtgctca 6600 tcctgctggc attctggagg gaaatacaac cgacagatgt atgtaaggcc aacgtgctca 6600 aatcttcata cagaaagatt tgaagtaata ttttaaccgc tagatgaaga gcaagcgcat 6660 aatcttcata cagaaagatt tgaagtaata ttttaaccgc tagatgaaga gcaagcgcat 6660 ggagcgacaa aatgaataaa gaacaatctg ctgatgatcc ctccgtggat ctgattcgtg 6720 ggagcgacaa aatgaataaa gaacaatctg ctgatgatcc ctccgtggat ctgattcgtg 6720 taaaaaatat gcttaatagc accatttcta tgagttaccc tgatgttgta attgcatgta 6780 taaaaaatat gcttaatagc accatttcta tgagttaccc tgatgttgta attgcatgta 6780 tagaacataa ggtgtctctg gaagcattca gagcaattga ggcagcgttg gtgaagcacg 6840 tagaacataa ggtgtctctg gaagcattca gagcaattga ggcagcgttg gtgaagcacg 6840 ataataatat gaaggattat tccctggtgg ttgactgatc accataactg ctaatcattc 6900 ataataatat gaaggattat tccctggtgg ttgactgatc accataactg ctaatcattc 6900 aaactattta gtctgtgaca gagccaacac gcagtctgtc actgtcagga aagtggtaaa 6960 aaactattta gtctgtgaca gagccaacac gcagtctgtc actgtcagga aagtggtaaa 6960 actgcaactc aattactgca atgccctcgt aattaagtga atttacaata tcgtcctgtt 7020 actgcaactc aattactgca atgccctcgt aattaagtga atttacaata tcgtcctgtt 7020 cggagggaag aacgcgggat gttcattctt catcactttt aattgatgta tatgctctct 7080 cggagggaag aacgcgggat gttcattctt catcactttt aattgatgta tatgctctct 7080 tttctgacgt tagtctccga cggcaggctt caatgaccca ggctgagaaa ttcccggacc 7140 tttctgacgt tagtctccga cggcaggctt caatgaccca ggctgagaaa ttcccggacc 7140 ctttttgctc aagagcgatg ttaatttgtt caatcatttg gttaggaaag cggatgttgc 7200 ctttttgctc aagagcgatg ttaatttgtt caatcatttg gttaggaaag cggatgttgc 7200 gggttgttgt tctgcgggtt ctgttcttcg ttgacatgag gttgccccgt attcagtgtc 7260 gggttgttgt tctgcgggtt ctgttcttcg ttgacatgag gttgccccgt attcagtgtc 7260 gctgatttgt attgtctgaa gttgttttta cgttaagttg atgcagatca attaatacga 7320 gctgatttgt attgtctgaa gttgttttta cgttaagttg atgcagatca attaatacga 7320 tacctgcgtc ataattgatt atttgacgtg gtttgatggc ctccacgcac gttgtgatat 7380 tacctgcgtc ataattgatt atttgacgtg gtttgatggc ctccacgcac gttgtgatat 7380 gtagatgata atcattatca ctttacgggt cctttccggt gatccgacag gttacggcct 7440 gtagatgata atcattatca ctttacgggt cctttccggt gatccgacag gttacggcct 7440 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa 7500 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa 7500 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 7560 ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcago 7560 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 7620 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 7620 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 7680 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 7680 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt 7740 cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt 7740 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 7800 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 7800 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt 7860 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt 7860 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 7920 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 7920 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt 7980 aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt 7980 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 8040 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 8040 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 8100 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 8100 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 8160 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 8160 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 8220 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 8220 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 8280 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 8280 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 8340 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 8340 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 8400 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 8400 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 8460 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 8460 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 8520 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 8520 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 8580 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 8580 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 8640 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 8640 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 8700 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 8700 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 8760 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 8760 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 8820 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 8820 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 8880 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 8880 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 8940 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 8940 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 9000 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 9000 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 9060 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 9060 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 9120 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 9120 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 9180 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 9180 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 9240 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 9240 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 9300 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 9300 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 9360 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 9360 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 9420 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 9420 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 9480 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 9480 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 9540 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag 9540 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 9600 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 9600 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 9660 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 9660 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag gctatgagaa agacgatagt agcgaacgac ctacaccgaa ctgagatacc tacagcgtga cagggtcgga agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 9720 9720 cccagcttgg ttcccgaagg gagaaaggcg gacaggtato cggtaagcgg tagtcctgtc cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 9780 9780 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 9840 agcgccacgc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta ggggcggagc 9840 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 9900 acaggagage acctctgact tgagcgtcga tttttgtgat gctcgtcagg ctggcctttt 9900 gggtttcgcc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 9960 9960 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 10020 10020 gctcacatgt cctgcaggca g gctcacatgt cctgcaggca g 10041 10041

<210> 9 <210> 9 <211> 10035 <211> 10035 <212> DNA <212> Artificial <213> DNA Sequence <213> Artificial Sequence <220> <223> <220> <223> pAAV_ATG5_C4‐CD28CD3z‐CAR.xdna <400> 9 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ctccatcact

<400> 9 ctgcgcgctc cctcagtgag cgagcgagcg cgcagagagg gagtggccaa cacatagact ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 ggtcgcccgg gcggcctaag cttgagcgga gttccaattg tactgtacag cttctgattt 120

aggggttcct agttaatgga ttatgtattt tacaaacact tttcaaatgt cctctgtcag aggggttcct gcggcctaag cttgagcgga gttccaattg tactgtacag cacatagact 180 180

tgcaggtgtg tgtttccctt ttctaagtgc atgtataatc ccagtttatg ctcaattgtc tgcaggtgtg agttaatgga ttatgtattt tacaaacact tttcaaatgt cttctgattt 240 240

tggtgtgctt ctgcctgttc actgaatagc ccataatagg caaagatatt ggagatgatg tggtgtgctt tgtttccctt ttctaagtgc atgtataatc ccagtttatg cctctgtcag 300 300

agatgacatc tttctttttg tgtctcaggt atgcattcta gtgtttacct tttaatgaag agatgacatc ctgcctgttc actgaatagc ccataatagg caaagatatt ctcaattgtc 360 360

gttactgttt gaagtgtatt tgtgatgagg aaatgcttct tctgtttcac agcctgctcc gttactgttt tttctttttg tgtctcaggt atgcattcta gtgtttacct ggagatgatg 420 420

tggaaaagta gctttccccc tcatctctct gagaggcagt gctgctcctc tcgctcactt tggaaaagta gaagtgtatt tgtgatgagg aaatgcttct tctgtttcac tttaatgaag 480 480

tctgcccttt agtcagatta ctggaggaag ctacttatta aggagcccat gcatctggca tctgcccttt gctttccccc tcatctctct gagaggcagt gctgctcctc agcctgctcc 540 540

agctacggaa agtcagatta ctggaggaag ctacttatta aggagcccat tcgctcactt 600 agctacggaa ggactgtttt tttgtttttg ttttcctctg tgcatcctca agagatctgg 600

tttgggtata aaaagcttct gacctcttct cttcctccca cagggcctcg atcccggccc tttgggtata ggactgtttt tttgtttttg ttttcctctg tgcatcctca gcatctggca 660 660

caggctcctc ggcagaggaa gtcttctaac atgcggtgac gtggaggaga acgccgccag caggctcctc aaaagcttct gacctcttct cttcctccca cagggcctcg agagatctgg 720 720

cggcggagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atcccggccc 780 cggcggagag ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc ggtccctgag 780 tatggcctta gccgggatcc cagctggtgg agtctggggg aggcttggta cagccagggc tatggcctta ccagtgaccg ccttgctcct gccgctggcc ttgctgctcc acgccgccag 840 840

gccgggatcc cagctggtgg agtctggggg aggcttggta cagccagggc ggtccctgag 900 actctcctgc acaacttctg gattcacttt tggtgattat gctatgatct gggcccgcca 960 096 ggctccaggg aaggggctgg agtgggtctc atccattagt agtagtagta gttacatata 1020 0201 the ctacgcagac tcagtgaagg gccgattcac catctccaga gacaacgcca agaactcact 1080 080I gtatctgcaa atgaacagcc tgagagccga ggacacggct gtgtattact gtgcgagaga 1140 acgatacgat ttttggagtg gaatggacgt ctggggcaaa gggaccacgg tcaccgtctc 1200 gagtggtgga ggcggttcag gcggaggtgg ctctggcggt agtgcacagt ctgccctgac 1260 The tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc accatctcct gcactggaac 1320 OZET cagcagtgat gttgggagtt ataaccttgt ctcctggtac caacagcacc caggcaaagc 1380 08EI ccccaaactc atgatttatg agggcagtaa gcggccctca ggggtttcta atcgcttctc 1440 tggctccaag tctggcaacg cggcctccct gacaatctct gggctccagg ctgaggacga 1500 00ST ggctgattat tactgccagt cctatgacag cagcctgagt gtggtattcg gcggagggac 1560 09ST caagctgacc gtcctaggtg ctagcaccac gacgccagcg ccgcgaccac caacaccggc 1620 The gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg 1680 089T gggcgcagtg cacacgaggg ggctggactt cgcctgtgat ttttgggtgc tggtggtggt 1740 tggtggagtc ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt 1800 008 gaggagtaag aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc 1860 098T cgggcccacc cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg 1920 ctccatcgat agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca 1980 086T gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa 2040 gagacgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg 2100 00I2 cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa 2160 09T2 aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac 2220 0222 caaggacacc tacgacgccc ttcacatgca ggccctgccc cctcgctgat aagcggccgc 2280 0822 e ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 2340 OTEL tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 2400 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 2460 credit gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgggtcga cgtggtacta 2520 ctcggtgcat gaatgtttcc ccaggctcat gttgggattt tgtttgaaat catctttatg 2580 0852 ggttttctta ctagaaatga tagttttaga gaagttcggc aatcttgtta ctggtactca 2640 agcttatata cacgtgagta atcttaactt tggaaatatc aaccgttttc tgtctagaaa 2700 00/2 aacatccaca gttaaaaacc aagtatggtc atagttttat ttttagaaga atttcatttg 2760 09/2

2777777777 7787077777 ttagaggtat tttttctgtt cttgttctgt aatataggtt aggataccat tttttttttc 2820 0282

caaacaagtt tttgctgttt taattacaac ttttaacctt cattggcttt gtaatagtat 2880 0882

the ataagagtat gtgagagtat aagagtatat ttggccaaat tgacaacaac tgggtaccct 2940 797 cgagcgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 3000 000E

actgaggccg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc 3060 090E

tgcaggggca gcttgaagga aatactaagg caaaggtact gcaagtgctc gcaacattcg 3120 OZIE

the cttatgcgga ttattgccgt agtgccgcga cgccgggggc aagatgcaga gattgccatg 3180 08IE

gtacaggccg tgcggttgat attgccaaaa cagagctgtg ggggagagtt gtcgagaaag 3240 credit agtgcggaag atgcaaaggc gtcggctatt caaggatgcc agcaagcgca gcatatcgcg 3300 00EE

ctgtgacgat gctaatccca aaccttaccc aacccacctg gtcacgcact gttaagccgc 3360 09EE

tgtatgacgc tctggtggtg caatgccaca aagaagagtc aatcgcagac aacattttga 3420

atgcggtcac acgttagcag catgattgcc acggatggca acatattaac ggcatgatat 3480 7874

the tgacttattg aataaaattg ggtaaatttg actcaacgat gggttaattc gctcgttgtg 3540

gtagtgagat gaaaagaggc ggcgcttact accgattccg cctagttggt cacttcgacg 3600 009E

tatcgtctgg aactccaacc atcgcaggca gagaggtctg caaaatgcaa tcccgaaaca 3660 099E

gttcgcaggt aatagttaga gcctgcataa cggtttcggg attttttata tctgcacaac 3720 OZLE

aggtaagagc attgagtcga taatcgtgaa gagtcggcga gcctggttag ccagtgctct 3780 08LE

the ttccgttgtg ctgaattaag cgaataccgg aagcagaacc ggatcaccaa atgcgtacag 3840 cheese gcgtcatcgc cgcccagcaa cagcacaacc caaactgagc cgtagccact gtctgtcctg 3900 006E

aattcattag taatagttac gctgcggcct tttacacatg accttcgtga aagcgggtgg 3960 096E

caggaggtcg cgctaacaac ctcctgccgt tttgcccgtg catatcggtc acgaacaaat 4020

ctgattacta aacacagtag cctggatttg ttctatcagt aatcgacctt attcctaatt 4080 0801

aaatagagca aatcccctta ttgggggtaa gacatgaaga tgccagaaaa acatgacctg 4140 ttggccgcca ttctcgcggc aaaggaacaa ggcatcgggg caatccttgc gtttgcaatg 4200

7 gcgtaccttc gcggcagata taatggcggt gcgtttacaa aaacagtaat cgacgcaacg 4260

the the e 7 atgtgcgcca ttatcgcctg gttcattcgt gaccttctcg acttcgccgg actaagtagc 4320 the the aatctcgctt atataacgag cgtgtttatc ggctacatcg gtactgactc gattggttcg 4380 08E cttatcaaac gcttcgctgc taaaaaagcc ggagtagaag atggtagaaa tcaataatca 4440

acgtaaggcg ttcctcgata tgctggcgtg gtcggaggga actgataacg gacgtcagaa 4500 00 aaccagaaat catggttatg acgtcattgt aggcggagag ctatttactg attactccga 4560

tcaccctcgc aaacttgtca cgctaaaccc aaaactcaaa tcaacaggcg ccggacgcta 4620

ccagcttctt tcccgttggt gggatgccta ccgcaagcag cttggcctga aagacttctc 4680 089/7

tccgaaaagt caggacgctg tggcattgca gcagattaag gagcgtggcg ctttacctat 4740

gattgatcgt ggtgatatcc gtcaggcaat cgaccgttgc agcaatatct gggcttcact 4800 008/

gccgggcgct ggttatggtc agttcgagca taaggctgac agcctgattg caaaattcaa 4860 098 -

agaagcgggc ggaacggtca gagagattga tgtatgagca gagtcaccgc gattatctcc 4920

gctctggtta tctgcatcat cgtctgcctg tcatgggctg ttaatcatta ccgtgataac 4980 086/

gccattacct acaaagccca gcgcgacaaa aatgccagag aactgaagct ggcgaacgcg 5040

gcaattactg acatgcagat gcgtcagcgt gatgttgctg cgctcgatgc aaaatacacg 5100 00IS

aaggagttag ctgatgctaa agctgaaaat gatgctctgc gtgatgatgt tgccgctggt 5160 09TS

cgtcgtcggt tgcacatcaa agcagtctgt cagtcagtgc gtgaagccac caccgcctcc 5220 0225

ggcgtggata atgcagcctc cccccgactg gcagacaccg ctgaacggga ttatttcacc 5280 0825

ctcagagaga ggctgatcac tatgcaaaaa caactggaag gaacccagaa gtatattaat 5340 ODES

gagcagtgca gatagagttg cccatatcga tgggcaactc atgcaattat tgtgagcaat 5400

acacacgcgc ttccagcgga gtataaatgc ctaaagtaat aaaaccgagc aatccattta 5460

the cgaatgtttg ctgggtttct gttttaacaa cattttctgc gccgccacaa attttggctg 5520

the catcgacagt tttcttctgc ccaattccag aaacgaagaa atgatgggtg atggtttcct 5580 0855

ttggtgctac tgctgccggt ttgttttgaa cagtaaacgt ctgttgagca catcctgtaa 5640

taagcagggc cagcgcagta gcgagtagca tttttttcat ggtgttattc ccgatgcttt 5700 00LS

ttgaagttcg cagaatcgta tgtgtagaaa attaaacaaa ccctaaacaa tgagttgaaa 5760 09/9

the tttcatattg ttaatattta ttaatgtatg tcaggtgcga tgaatcgtca ttgtattccc 5820 tttcatattg ttaatattta ttaatgtatg tcaggtgcga tgaatcgtca ttgtattccc 5820 ggattaacta tgtccacagc cctgacgggg aacttctctg cgggagtgtc cgggaataat 5880 ggattaacta tgtccacagc cctgacgggg aacttctctg cgggagtgtc cgggaataat 5880 taaaacgatg cacacagggt ttagcgcgta cacgtattgc attatgccaa cgccccggtg 5940 taaaacgatg cacacagggt ttagcgcgta cacgtattgc attatgccaa cgccccggtg 5940 ctgacacgga agaaaccgga cgttatgatt tagcgtggaa agatttgtgt agtgttctga 6000 ctgacacgga agaaaccgga cgttatgatt tagcgtggaa agatttgtgt agtgttctga 6000 atgctctcag taaatagtaa tgaattatca aaggtatagt aatatctttt atgttcatgg 6060 atgctctcag taaatagtaa tgaattatca aaggtatagt aatatctttt atgttcatgg 6060 atatttgtaa cccatcggaa aactcctgct ttagcaagat tttccctgta ttgctgaaat 6120 atatttgtaa cccatcggaa aactcctgct ttagcaagat tttccctgta ttgctgaaat 6120 gtgatttctc ttgatttcaa cctatcatag gacgtttcta taagatgcgt gtttcttgag 6180 gtgatttctc ttgatttcaa cctatcatag gacgtttcta taagatgcgt gtttcttgag 6180 aatttaacat ttacaacctt tttaagtcct tttattaaca cggtgttatc gttttctaac 6240 aatttaacat ttacaacctt tttaagtcct tttattaaca cggtgttatc gttttctaac 6240 acgatgtgaa tattatctgt ggctagatag taaatataat gtgagacgtt gtgacgtttt 6300 acgatgtgaa tattatctgt ggctagatag taaatataat gtgagacgtt gtgacgtttt 6300 agttcagaat aaaacaattc acagtctaaa tcttttcgca cttgatcgaa tatttcttta 6360 agttcagaat aaaacaattc acagtctaaa tcttttcgca cttgatcgaa tatttcttta 6360 aaaatggcaa cctgagccat tggtaaaacc ttccatgtga tacgagggcg cgtagtttgc 6420 aaaatggcaa cctgagccat tggtaaaacc ttccatgtga tacgagggcg cgtagtttgc 6420 attatcgttt ttatcgtttc aatctggtct gacctccttg tgttttgttg atgatttatg 6480 attatcgttt ttatcgtttc aatctggtct gacctccttg tgttttgttg atgatttatg 6480 tcaaatatta ggaatgtttt cacttaatag tattggttgc gtaacaaagt gcggtcctgc 6540 tcaaatatta ggaatgtttt cacttaatag tattggttgc gtaacaaagt gcggtcctgc 6540 tggcattctg gagggaaata caaccgacag atgtatgtaa ggccaacgtg ctcaaatctt 6600 tggcattctg gagggaaata caaccgacag atgtatgtaa ggccaacgtg ctcaaatctt 6600 catacagaaa gatttgaagt aatattttaa ccgctagatg aagagcaagc gcatggagcg 6660 catacagaaa gatttgaagt aatattttaa ccgctagatg aagagcaage gcatggagcg 6660 acaaaatgaa taaagaacaa tctgctgatg atccctccgt ggatctgatt cgtgtaaaaa 6720 acaaaatgaa taaagaacaa tctgctgatg atccctccgt ggatctgatt cgtgtaaaaa 6720 atatgcttaa tagcaccatt tctatgagtt accctgatgt tgtaattgca tgtatagaac 6780 atatgcttaa tagcaccatt tctatgagtt accctgatgt tgtaattgca tgtatagaac 6780 ataaggtgtc tctggaagca ttcagagcaa ttgaggcagc gttggtgaag cacgataata 6840 ataaggtgtc tctggaagca ttcagagcaa ttgaggcagc gttggtgaag cacgataata 6840 atatgaagga ttattccctg gtggttgact gatcaccata actgctaatc attcaaacta 6900 atatgaagga ttattccctg gtggttgact gatcaccata actgctaatc attcaaacta 6900 tttagtctgt gacagagcca acacgcagtc tgtcactgtc aggaaagtgg taaaactgca 6960 tttagtctgt gacagagcca acacgcagtc tgtcactgtc aggaaagtgg taaaactgca 6960 actcaattac tgcaatgccc tcgtaattaa gtgaatttac aatatcgtcc tgttcggagg 7020 actcaattac tgcaatgccc tcgtaattaa gtgaatttac aatatcgtcc tgttcggagg 7020 gaagaacgcg ggatgttcat tcttcatcac ttttaattga tgtatatgct ctcttttctg 7080 gaagaacgcg ggatgttcat tcttcatcac ttttaattga tgtatatgct ctcttttctg 7080 acgttagtct ccgacggcag gcttcaatga cccaggctga gaaattcccg gacccttttt 7140 acgttagtct ccgacggcag gcttcaatga cccaggctga gaaattcccg gacccttttt 7140 gctcaagagc gatgttaatt tgttcaatca tttggttagg aaagcggatg ttgcgggttg 7200 gctcaagagc gatgttaatt tgttcaatca tttggttagg aaagcggatg ttgcgggttg 7200 ttgttctgcg ggttctgttc ttcgttgaca tgaggttgcc ccgtattcag tgtcgctgat 7260 ttgttctgcg ggttctgttc ttcgttgaca tgaggttgcc ccgtattcag tgtcgctgat 7260 ttgtattgtc tgaagttgtt tttacgttaa gttgatgcag atcaattaat acgatacctg 7320 ttgtattgtc tgaagttgtt tttacgttaa gttgatgcag atcaattaat acgatacctg 7320 cgtcataatt gattatttga cgtggtttga tggcctccac gcacgttgtg atatgtagat 7380 cgtcataatt gattatttga cgtggtttga tggcctccac gcacgttgtg atatgtagat 7380 gataatcatt atcactttac gggtcctttc cggtgatccg acaggttacg gcctgatgcg 7440 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 7500 0052 the tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 7560 09SL the gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 7620 0292 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 7680 089L the agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 7740 DILL ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 7800 008L the ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 7860 098L taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 7920 0262 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 7980 086L tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 8040 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 8100 0018 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 8160 2777788e8e 09t8 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 8220 0228 the acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 8280 0828 the atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 8340 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 8400 e the cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 8460 799 the gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 8520 0258 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 8580 0898 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 8640 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 8700 00/8 ee ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 8760 09/8 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 8820 0288 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 8880 0888 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 8940 7968 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 9000 0006 the cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 9060 9060 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 9120 9120 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatogc tgagataggt tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 9180 9180 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 9240 9240 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 9300 9300 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 9360 9360 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 9420 9420 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 9480 9480 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 9540 9540 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 9600 9600 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 9660 9660 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccago tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 9720 9720 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 9780 9780 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 9840 9840 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 9900 9900 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 9960 9960 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 10020 10020 atgtcctgca ggcag 10035 atgtcctgca ggcag 10035

<210> 10 <210> 10 <211> 10782 <211> 10782 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> pAAV_AAVS1_C4‐CD27CD3z‐CAR.xdna <223> pAAV_AAVS1_C4-CD27CD3z-CAR.xdna

<400> 10 <400> 10 ctgcgcgctc gctcgctcad tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 120

aggggttcct gcggcctaag cttgagcgga gttccaattg tactgtacag ccctttgctt aggggttcct gcggcctaag cttgagcgga gttccaattg tactgtacag ccctttgctt 180 180

tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct 240 gggtcacctc tccgtcttcc gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc 300 300 tccactccct tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag 360 360 cacttccttc ccgtgtctgg cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg 420 420 gtcctctccg tcgctgggtt gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt 480 480 cccttttcct cccttttcct tctccttctg gggcctgtgc catctctcgt ttcttaggat ggccttctcc 540 540 gacggatgtc gacggatgtc tcccttgcgt cccgcctccc cttcttgtag gcctgcatca tcaccgtttt 600 600 tctggacaac tggctttagc tctggacaac cccaaagtac cccgtctccc tggctttagc cacctctcca tcctcttgct 660 660 ttctttgcct cctttcattt ttctttgcct ggacaccccg ttctcctgtg gattcgggtc acctctcact cctttcattt 720 720 gggcagctcc gcccccctgtc gggcagctcc cctacccccc ttacctctct agtctgtgct agctcttcca gccccctgtc 780 780 atggcatctt atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc cgggccccta 840 840 tgtccacttc gagctgggac tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc gagctgggac 900 900 caccttatat tatctgtccc caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt tatctgtccc 960 960 ctccacccca agcttctgac ggcctcgaga ctccacccca cagtggggca agcttctgac ctcttctctt cctcccacag ggcctcgaga 1020 1020 gatctggcgg gaggagaatc gatctggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc 1080 1080 ccggccctat ccggccctat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 1140 1140 ccgccaggcc ctggtggagt ccagggcggt ccgccaggcc gggatcccag ctggtggagt ctgggggagg cttggtacag ccagggcggt 1200 1200 ccctgagact atgatctggg ccctgagact ctcctgcaca acttctggat tcacttttgg tgattatgct atgatctggg 1260 1260 cccgccaggc agtagtagtt cccgccaggc tccagggaag gggctggagt gggtctcatc cattagtagt agtagtagtt 1320 1320 acatatacta aacgccaaga acatatacta cgcagactca gtgaagggcc gattcaccat ctccagagac aacgccaaga 1380 1380 actcactgta tctgcaaatg tattactgtg actcactgta tctgcaaatg aacagcctga gagccgagga cacggctgtg tattactgtg 1440 1440 cgagagaacg tggagtggaa cgagagaacg atacgatttt tggagtggaa tggacgtctg gggcaaaggg accacggtca 1500 1500 ccgtctcgag tggtggaggc gcacagtctg ccgtctcgag tggtggaggc ggttcaggcg gaggtggctc tggcggtagt gcacagtctg 1560 1560 ccctgactca ccctgactca gcctgcctcc gtgtctgggt ctcctggaca gtcgatcacc atctcctgca 1620 1620 ctggaaccag accttgtctc ctggaaccag cagtgatgtt gggagttata accttgtctc ctggtaccaa cagcacccag 1680 1680 gcaaagcccc caaactcatg gcagtaagcg gtttctaatc gcaaagcccc caaactcatg atttatgagg gcagtaagcg gccctcaggg gtttctaatc 1740 1740 gcttctctgg ggcaacgcgg aatctctggg ctccaggctg gcttctctgg ctccaagtct ggcaacgcgg cctccctgac aatctctggg ctccaggctg 1800 1800 aggacgaggc tgattattac tgccagtcct atgacagcag cctgagtgtg gtattcggcg aggacgaggc tgattattac tgccagtcct atgacagcag cctgagtgtg gtattcggcg 1860 gagggaccaa gctgaccgtc ctaggtgcta gcaccacgac gccagcgccg cgaccaccaa 1920 026T caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg tgccggccag 1980 086T

999999,995

e cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc tacatctggg 2040

cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc ctttactgcc 2100 0012

aacgaaggaa atatagatca aacaaaggag aaagtcctgt ggagcctgca gagccttgtc 2160 09T2

gttacagctg ccccagggag gaggagggca gcaccatccc catccaggag gattaccgaa 2220 0222

aaccggagcc tgcctgctcc cccagagtga agttcagcag gagcgcagac gcccccgcgt 2280 0822

e accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga gaggagtacg 2340 OTEL

atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg agaaggaaga 2400

the e accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag gcctacagtg 2460

agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt taccagggtc 2520 0252

tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg ccccctcgct 2580 0852

e e gataagcggc cgcctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 2640 797 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 2700 00/2

gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 2760 9997888918 09/2

aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgggt 2820 0282

cgacagtact aagctttact agggacagga ttggtgacag aaaagcccca tccttaggcc 2880 0887

ee tcctccttcc tagtctcctg atattgggtc taacccccac ctcctgttag gcagattcct 2940

tatctggtga cacaccccca tttcctggag ccatctctct ccttgccaga acctctaagg 3000 000E

tttgcttacg atggagccag agaggatcct gggagggaga gcttggcagg gggtgggagg 3060 090E

gaaggggggg atgcgtgacc tgcccggttc tcagtggcca ccctgcgcta ccctctccca 3120 9999999ee8 OTTE

e gaacctgagc tgctctgacg cggctgtctg gtgcgtttca ctgatcctgg tgctgcagct 3180

the 08TE

tccttacact tcccaagagg agaagcagtt tggaaaaaca aaatcagaat aagttggtcc 3240

tgagttctaa ctttggctct tcacctttct agtccccaat ttatattgtt cctccgtgcg 3300 00EE

tcagttttac ctgtgagata aggccagtag ccagccccgt cctggcaggg ctgtggtgag 3360 09EE

gaggggggtg tccgtgtgga aaactccctt tgtgagaatg gtgcgtccta ggtgttcacc 3420 91999999e8

aggtcgtggc cgcctctact ccctttctct ttctccatcc ttctttcctt aaagagtccc 3480 cagtgctatc tgggacatat tcctccgccc agagcagggt cccgcttccc taaggccctg 3540 ctctgggctt ctgggtttga gtccttggca agcccaggag aggcgctcag gcttccctgt 3600 009E cccccttcct cgtccaccat ctcatgcccc tggctctcct gccccttccc tacaggggtt 3660 099E cctggctctg ctctaagggg gtaccctcga gcgcaggaac ccctagtgat ggagttggcc 3720 OZLE actccctctc tgcgcgctcg ctcgctcact gaggccgccc gggctttgcc cgggcggcct 3780 08LE cagtgagcga gcgagcgcgc agctgcctgc aggggcagct tgaaggaaat actaaggcaa 3840 aggtactgca agtgctcgca acattcgctt atgcggatta ttgccgtagt gccgcgacgc 3900 006E cgggggcaag atgcagagat tgccatggta caggccgtgc ggttgatatt gccaaaacag 3960 0968 agctgtgggg gagagttgtc gagaaagagt gcggaagatg caaaggcgtc ggctattcaa 4020 0201 ggatgccagc aagcgcagca tatcgcgctg tgacgatgct aatcccaaac cttacccaac 4080 0801 ccacctggtc acgcactgtt aagccgctgt atgacgctct ggtggtgcaa tgccacaaag 4140 aagagtcaat cgcagacaac attttgaatg cggtcacacg ttagcagcat gattgccacg 4200 the thethe gatggcaaca tattaacggc atgatattga cttattgaat aaaattgggt aaatttgact 4260 the caacgatggg ttaattcgct cgttgtggta gtgagatgaa aagaggcggc gcttactacc 4320 gattccgcct agttggtcac ttcgacgtat cgtctggaac tccaaccatc gcaggcagag 4380 08ED e aggtctgcaa aatgcaatcc cgaaacagtt cgcaggtaat agttagagcc tgcataacgg 4440 tttcgggatt ttttatatct gcacaacagg taagagcatt gagtcgataa tcgtgaagag 4500 tcggcgagcc tggttagcca gtgctctttc cgttgtgctg aattaagcga ataccggaag 4560 09 cagaaccgga tcaccaaatg cgtacaggcg tcatcgccgc ccagcaacag cacaacccaa 4620

7 actgagccgt agccactgtc tgtcctgaat tcattagtaa tagttacgct gcggcctttt 4680 089/7

e acacatgacc ttcgtgaaag cgggtggcag gaggtcgcgc taacaacctc ctgccgtttt 4740

gcccgtgcat atcggtcacg aacaaatctg attactaaac acagtagcct ggatttgttc 4800 008/7

tatcagtaat cgaccttatt cctaattaaa tagagcaaat ccccttattg ggggtaagac 4860 098t

atgaagatgc cagaaaaaca tgacctgttg gccgccattc tcgcggcaaa ggaacaaggc 4920

atcggggcaa tccttgcgtt tgcaatggcg taccttcgcg gcagatataa tggcggtgcg 4980 086t

tttacaaaaa cagtaatcga cgcaacgatg tgcgccatta tcgcctggtt cattcgtgac 5040

cttctcgact tcgccggact aagtagcaat ctcgcttata taacgagcgt gtttatcggc 5100 00IS e the the tacatcggta ctgactcgat tggttcgctt atcaaacgct tcgctgctaa aaaagccgga 5160 09TS gtagaagatg gtagaaatca ataatcaacg taaggcgttc ctcgatatgc tggcgtggtc 5220 0225 ggagggaact gataacggac gtcagaaaac cagaaatcat ggttatgacg tcattgtagg 5280 0825 cggagagcta tttactgatt actccgatca ccctcgcaaa cttgtcacgc taaacccaaa 5340 OTES actcaaatca acaggcgccg gacgctacca gcttctttcc cgttggtggg atgcctaccg 5400 caagcagctt ggcctgaaag acttctctcc gaaaagtcag gacgctgtgg cattgcagca 5460 gattaaggag cgtggcgctt tacctatgat tgatcgtggt gatatccgtc aggcaatcga 5520 0255 ccgttgcagc aatatctggg cttcactgcc gggcgctggt tatggtcagt tcgagcataa 5580 0855 ggctgacagc ctgattgcaa aattcaaaga agcgggcgga acggtcagag agattgatgt 5640 atgagcagag tcaccgcgat tatctccgct ctggttatct gcatcatcgt ctgcctgtca 5700 00/5 tgggctgtta atcattaccg tgataacgcc attacctaca aagcccagcg cgacaaaaat 5760 09/9 gccagagaac tgaagctggc gaacgcggca attactgaca tgcagatgcg tcagcgtgat 5820 0289 the The gttgctgcgc tcgatgcaaa atacacgaag gagttagctg atgctaaagc tgaaaatgat 5880 088S gctctgcgtg atgatgttgc cgctggtcgt cgtcggttgc acatcaaagc agtctgtcag 5940 the tcagtgcgtg aagccaccac cgcctccggc gtggataatg cagcctcccc ccgactggca 6000 0009 gacaccgctg aacgggatta tttcaccctc agagagaggc tgatcactat gcaaaaacaa 6060 0909 ctggaaggaa cccagaagta tattaatgag cagtgcagat agagttgccc atatcgatgg 6120 0219 e gcaactcatg caattattgt gagcaataca cacgcgcttc cagcggagta taaatgccta 6180 08t9 aagtaataaa accgagcaat ccatttacga atgtttgctg ggtttctgtt ttaacaacat 6240 the tttctgcgcc gccacaaatt ttggctgcat cgacagtttt cttctgccca attccagaaa 6300 9777007778 the 00E9 cgaagaaatg atgggtgatg gtttcctttg gtgctactgc tgccggtttg ttttgaacag 6360 09E9 taaacgtctg ttgagcacat cctgtaataa gcagggccag cgcagtagcg agtagcattt 6420 declare 977777087e ttttcatggt gttattcccg atgctttttg aagttcgcag aatcgtatgt gtagaaaatt 6480 7879 aaacaaaccc taaacaatga gttgaaattt catattgtta atatttatta atgtatgtca 6540 ggtgcgatga atcgtcattg tattcccgga ttaactatgt ccacagccct gacggggaac 6600 0099 ttctctgcgg gagtgtccgg gaataattaa aacgatgcac acagggttta gcgcgtacac 6660 0999 gtattgcatt atgccaacgc cccggtgctg acacggaaga aaccggacgt tatgatttag 6720 0229 e the cgtggaaaga tttgtgtagt gttctgaatg ctctcagtaa atagtaatga attatcaaag 6780 00 bo gtatagtaat atcttttatg ttcatggata tttgtaaccc atcggaaaac tcctgcttta 6840 gcaagatttt ccctgtattg ctgaaatgtg atttctcttg atttcaacct atcataggac 6900 gtttctataa gatgcgtgtt tcttgagaat ttaacattta caaccttttt aagtcctttt 6960 attaacacgg tgttatcgtt ttctaacacg atgtgaatat tatctgtggc tagatagtaa 7020 atataatgtg agacgttgtg acgttttagt tcagaataaa acaattcaca gtctaaatct 7080 tttcgcactt gatcgaatat ttctttaaaa atggcaacct gagccattgg taaaaccttc 7140 00 catgtgatac gagggcgcgt agtttgcatt atcgttttta tcgtttcaat ctggtctgac 7200 ctccttgtgt tttgttgatg atttatgtca aatattagga atgttttcac ttaatagtat 7260 tggttgcgta acaaagtgcg gtcctgctgg cattctggag ggaaatacaa ccgacagatg 7320 00 tatgtaaggc caacgtgctc aaatcttcat acagaaagat ttgaagtaat attttaaccg 7380 bo ctagatgaag agcaagcgca tggagcgaca aaatgaataa agaacaatct gctgatgatc 7440 cctccgtgga tctgattcgt gtaaaaaata tgcttaatag caccatttct atgagttacc 7500 ctgatgttgt aattgcatgt atagaacata aggtgtctct ggaagcattc agagcaattg 7560 00 aggcagcgtt ggtgaagcac gataataata tgaaggatta ttccctggtg gttgactgat 7620 00 caccataact gctaatcatt caaactattt agtctgtgac agagccaaca cgcagtctgt 7680 cactgtcagg aaagtggtaa aactgcaact caattactgc aatgccctcg taattaagtg 7740 00 aatttacaat atcgtcctgt tcggagggaa gaacgcggga tgttcattct tcatcacttt 7800 taattgatgt atatgctctc ttttctgacg ttagtctccg acggcaggct tcaatgaccc 7860 aggctgagaa attcccggac cctttttgct caagagcgat gttaatttgt tcaatcattt 7920 00 ggttaggaaa gcggatgttg cgggttgttg ttctgcgggt tctgttcttc gttgacatga 7980 ggttgccccg tattcagtgt cgctgatttg tattgtctga agttgttttt acgttaagtt 8040 gatgcagatc aattaatacg atacctgcgt cataattgat tatttgacgt ggtttgatgg 8100 cctccacgca cgttgtgata tgtagatgat aatcattatc actttacggg tcctttccgg 8160 00 tgatccgaca ggttacggcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 8220 acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 8280 00 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 8340 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 8400 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 8400 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 8460 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 8460 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 8520 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 8520 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 8580 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 8580 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 8640 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta 8640 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 8700 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca 8700 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 8760 attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga 8760 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 8820 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac 8820 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 8880 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg 8880 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 8940 aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 8940 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 9000 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 9000 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 9060 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 9060 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 9120 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 9120 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 9180 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 9180 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 9240 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 9240 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 9300 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 9300 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 9360 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt 9360 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 9420 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 9420 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 9480 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 9480 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 9540 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 9540 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 9600 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 9600 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 9660 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa 9660 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 9720 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 9720 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 9780 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 9780 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 9840 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 9840 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 9900 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 9900 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 9960 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 9960 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 10020 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 10020 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 10080 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 10080 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 10140 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 10140 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 10200 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 10200 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 10260 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagago gcagatacca 10260 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 10320 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 10320 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 10380 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 10380 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 10440 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 10440 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 10500 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatad 10500 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 10560 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggo ggacaggtat 10560 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 10620 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 10620 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 10680 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 10680 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 10740 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 10740 ctggcctttt gctggccttt tgctcacatg tcctgcaggc ag 10782 ctggcctttt gctggccttt tgctcacatg tcctgcaggo ag 10782

<210> 11 <210> 11 <211> 10776 <211> 10776 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> pAAV_AAVS1_C4‐CD28CD3z‐CAR.xdna <223> pAAV_AAVS1_C4-CD28CD3z-CAR.xdna

<400> 11 <400> 11 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct gcggcctaag cttgagcgga gttccaattg tactgtacag ccctttgctt 180 aggggttcct gcggcctaag cttgagcgga gttccaattg tactgtacag ccctttgctt 180

tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct 240 tctctgacca gcattctctc ccctgggcct gtgccgcttt ctgtctgcag cttgtggcct 240

gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc 300 gggtcacctc tacggctggc ccagatcctt ccctgccgcc tccttcaggt tccgtcttcc 300

tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag 360 tccactccct cttccccttg ctctctgctg tgttgctgcc caaggatgct ctttccggag 360

cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg 420 cacttccttc tcggcgctgc accacgtgat gtcctctgag cggatcctcc ccgtgtctgg 420

gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt 480 gtcctctccg ggcatctctc ctccctcacc caaccccatg ccgtcttcac tcgctgggtt 480 cccttttcct tctccttctg gggcctgtgc catctctcgt ttcttaggat ggccttctcc 540 540 gacggatgtc tcccttgcgt cccgcctccc cttcttgtag gcctgcatca tcaccgtttt 600 600 tctggacaac cccaaagtac cccgtctccc tggctttagc cacctctcca tcctcttgct 660 660 ttctttgcct ggacaccccg ttctcctgtg gattcgggtc acctctcact cctttcattt 720 gggcagctcc cctacccccc ttacctctct agtctgtgct agctcttcca gccccctgtc 780 780 atggcatctt atggcatctt ccaggggtcc gagagctcag ctagtcttct tcctccaacc cgggccccta 840 840 tgtccacttc aggacagcat gtttgctgcc tccagggatc ctgtgtcccc gagctgggac 900 caccttatat tcccagggcc ggttaatgtg gctctggttc tgggtacttt tatctgtccc 960 960 ctccacccca cagtggggca agcttctgac ctcttctctt cctcccacag ggcctcgaga 1020 1020 gatctggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc 1080 ccggccctat ggccttacca gtgaccgcct tgctcctgcc gctggccttg ctgctccacg 1140 1140 ccgccaggcc gggatcccag ctggtggagt ctgggggagg cttggtacag ccagggcggt 1200 ccctgagact ctcctgcaca acttctggat tcacttttgg tgattatgct atgatctggg 1260 cccgccaggc tccagggaag gggctggagt gggtctcatc cattagtagt agtagtagtt 1320 1320 acatatacta cgcagactca gtgaagggcc gattcaccat ctccagagac aacgccaaga 1380 1380 actcactgta tctgcaaatg aacagcctga gagccgagga cacggctgtg tattactgtg 1440 1440 cgagagaacg atacgatttt tggagtggaa tggacgtctg gggcaaaggg accacggtca 1500 1500 ccgtctcgag tggtggaggc ggttcaggcg gaggtggctc tggcggtagt gcacagtctg 1560 1560 ccctgactca gcctgcctcc gtgtctgggt ctcctggaca gtcgatcacc atctcctgca 1620 ctggaaccag cagtgatgtt gggagttata accttgtctc ctggtaccaa cagcacccag 1680

1740 gcaaagcccc caaactcatg atttatgagg gcagtaagcg gccctcaggg gtttctaatc 1740

gcttctctgg ctccaagtct ggcaacgcgg cctccctgac aatctctggg ctccaggctg 1800

aggacgaggc tgattattac tgccagtcct atgacagcag cctgagtgtg gtattcggcg 1860 1860

gagggaccaa gctgaccgtc ctaggtgcta gcaccacgac gccagcgccg cgaccaccaa 1920 1920

caccggcgcc caccatcgcg tcgcagcccc tgtccctgcg cccagaggcg tgccggccag 1980 cggcgggggg cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatttt tgggtgctgg 2040

tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc tttattattt 2100 tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac atgactcccc 2160 tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac atgactcccc 2160 gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc gacttcgcag 2220 gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc gacttcgcag 2220 cctatcgctc catcgataga gtgaagttca gcaggagcgc agacgccccc gcgtaccagc 2280 cctatcgctc catcgataga gtgaagttca gcaggagcgc agacgccccc gcgtaccago 2280 agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag tacgatgttt 2340 agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag tacgatgttt 2340 tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg aagaaccctc 2400 tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg aagaaccctc 2400 aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 2460 aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac agtgagattg 2460 ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 2520 ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag ggtctcagta 2520 cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct cgctgataag 2580 cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct cgctgataag 2580 cggccgcctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2640 cggccgcctg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 2640 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2700 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 2700 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 2760 cattgtctga gtaggtgtca ttctattctg gggggtggggg tggggcagga cagcaagggg 2760 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat gggtcgacag 2820 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat gggtcgacag 2820 tactaagctt tactagggac aggattggtg acagaaaagc cccatcctta ggcctcctcc 2880 tactaagctt tactagggac aggattggtg acagaaaagc cccatcctta ggcctcctcc 2880 ttcctagtct cctgatattg ggtctaaccc ccacctcctg ttaggcagat tccttatctg 2940 ttcctagtct cctgatattg ggtctaaccc ccacctcctg ttaggcagat tccttatctg 2940 gtgacacacc cccatttcct ggagccatct ctctccttgc cagaacctct aaggtttgct 3000 gtgacacacc cccatttcct ggagccatct ctctccttgc cagaacctct aaggtttgct 3000 tacgatggag ccagagagga tcctgggagg gagagcttgg cagggggtgg gagggaaggg 3060 tacgatggag ccagagagga tcctgggagg gagagcttgg cagggggtgg gagggaaggg 3060 ggggatgcgt gacctgcccg gttctcagtg gccaccctgc gctaccctct cccagaacct 3120 ggggatgcgt gacctgcccg gttctcagtg gccaccctgc gctaccctct cccagaacct 3120 gagctgctct gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc agcttcctta 3180 gagctgctct gacgcggctg tctggtgcgt ttcactgatc ctggtgctgc agcttcctta 3180 cacttcccaa gaggagaagc agtttggaaa aacaaaatca gaataagttg gtcctgagtt 3240 cacttcccaa gaggagaage agtttggaaa aacaaaatca gaataagttg gtcctgagtt 3240 ctaactttgg ctcttcacct ttctagtccc caatttatat tgttcctccg tgcgtcagtt 3300 ctaactttgg ctcttcacct ttctagtccc caatttatat tgttcctccg tgcgtcagtt 3300 ttacctgtga gataaggcca gtagccagcc ccgtcctggc agggctgtgg tgaggagggg 3360 ttacctgtga gataaggcca gtagccagcc ccgtcctggc agggctgtgg tgaggagggg 3360 ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt caccaggtcg 3420 ggtgtccgtg tggaaaactc cctttgtgag aatggtgcgt cctaggtgtt caccaggtcg 3420 tggccgcctc tactcccttt ctctttctcc atccttcttt ccttaaagag tccccagtgc 3480 tggccgcctc tactcccttt ctctttctcc atccttcttt ccttaaagag tccccagtgc 3480 tatctgggac atattcctcc gcccagagca gggtcccgct tccctaaggc cctgctctgg 3540 tatctgggac atattcctcc gcccagagca gggtcccgct tccctaaggc cctgctctgg 3540 gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc ctgtccccct 3600 gcttctgggt ttgagtcctt ggcaagccca ggagaggcgc tcaggcttcc ctgtccccct 3600 tcctcgtcca ccatctcatg cccctggctc tcctgcccct tccctacagg ggttcctggc 3660 tcctcgtcca ccatctcatg cccctggctc tcctgcccct tccctacagg ggttcctggc 3660 tctgctctaa gggggtaccc tcgagcgcag gaacccctag tgatggagtt ggccactccc 3720 tctgctctaa gggggtaccc tcgagcgcag gaacccctag tgatggagtt ggccactccc 3720 tctctgcgcg ctcgctcgct cactgaggcc gcccgggctt tgcccgggcg gcctcagtga 3780 08LE gcgagcgagc gcgcagctgc ctgcaggggc agcttgaagg aaatactaag gcaaaggtac 3840 tgcaagtgct cgcaacattc gcttatgcgg attattgccg tagtgccgcg acgccggggg 3900 006E the caagatgcag agattgccat ggtacaggcc gtgcggttga tattgccaaa acagagctgt 3960 0968 gggggagagt tgtcgagaaa gagtgcggaa gatgcaaagg cgtcggctat tcaaggatgc 4020 cagcaagcgc agcatatcgc gctgtgacga tgctaatccc aaaccttacc caacccacct 4080 080/ ggtcacgcac tgttaagccg ctgtatgacg ctctggtggt gcaatgccac aaagaagagt 4140 caatcgcaga caacattttg aatgcggtca cacgttagca gcatgattgc cacggatggc 4200 aacatattaa cggcatgata ttgacttatt gaataaaatt gggtaaattt gactcaacga 4260 tgggttaatt cgctcgttgt ggtagtgaga tgaaaagagg cggcgcttac taccgattcc 4320 OZED gcctagttgg tcacttcgac gtatcgtctg gaactccaac catcgcaggc agagaggtct 4380 08E gcaaaatgca atcccgaaac agttcgcagg taatagttag agcctgcata acggtttcgg 4440 gattttttat atctgcacaa caggtaagag cattgagtcg ataatcgtga agagtcggcg 4500 the the agcctggtta gccagtgctc tttccgttgt gctgaattaa gcgaataccg gaagcagaac 4560 the been cggatcacca aatgcgtaca ggcgtcatcg ccgcccagca acagcacaac ccaaactgag 4620 ccgtagccac tgtctgtcct gaattcatta gtaatagtta cgctgcggcc ttttacacat 4680 089/ gaccttcgtg aaagcgggtg gcaggaggtc gcgctaacaa cctcctgccg ttttgcccgt 4740 The gcatatcggt cacgaacaaa tctgattact aaacacagta gcctggattt gttctatcag 4800 008/ taatcgacct tattcctaat taaatagagc aaatcccctt attgggggta agacatgaag 4860 098t atgccagaaa aacatgacct gttggccgcc attctcgcgg caaaggaaca aggcatcggg 4920 the gcaatccttg cgtttgcaat ggcgtacctt cgcggcagat ataatggcgg tgcgtttaca 4980 086t the aaaacagtaa tcgacgcaac gatgtgcgcc attatcgcct ggttcattcg tgaccttctc 5040 the gacttcgccg gactaagtag caatctcgct tatataacga gcgtgtttat cggctacatc 5100 00IS ggtactgact cgattggttc gcttatcaaa cgcttcgctg ctaaaaaagc cggagtagaa 5160 09TS gatggtagaa atcaataatc aacgtaaggc gttcctcgat atgctggcgt ggtcggaggg 5220 0225 aactgataac ggacgtcaga aaaccagaaa tcatggttat gacgtcattg taggcggaga 5280 0825 eee the gctatttact gattactccg atcaccctcg caaacttgtc acgctaaacc caaaactcaa 5340 OTES atcaacaggc gccggacgct accagcttct ttcccgttgg tgggatgcct accgcaagca 5400 gcttggcctg aaagacttct ctccgaaaag tcaggacgct gtggcattgc agcagattaa 5460 ggagcgtggc gctttaccta tgattgatcg tggtgatatc cgtcaggcaa tcgaccgttg 5520 0255 cagcaatatc tgggcttcac tgccgggcgc tggttatggt cagttcgagc ataaggctga 5580 0855 cagcctgatt gcaaaattca aagaagcggg cggaacggtc agagagattg atgtatgagc 5640 agagtcaccg cgattatctc cgctctggtt atctgcatca tcgtctgcct gtcatgggct 5700 00LS gttaatcatt accgtgataa cgccattacc tacaaagccc agcgcgacaa aaatgccaga 5760 09/9 gaactgaagc tggcgaacgc ggcaattact gacatgcaga tgcgtcagcg tgatgttgct 5820 0789 gcgctcgatg caaaatacac gaaggagtta gctgatgcta aagctgaaaa tgatgctctg 5880 088S cgtgatgatg ttgccgctgg tcgtcgtcgg ttgcacatca aagcagtctg tcagtcagtg 5940 cgtgaagcca ccaccgcctc cggcgtggat aatgcagcct ccccccgact ggcagacacc 6000 0009 gctgaacggg attatttcac cctcagagag aggctgatca ctatgcaaaa acaactggaa 6060 0909 ggaacccaga agtatattaa tgagcagtgc agatagagtt gcccatatcg atgggcaact 6120 0219 catgcaatta ttgtgagcaa tacacacgcg cttccagcgg agtataaatg cctaaagtaa 6180 08t9 taaaaccgag caatccattt acgaatgttt gctgggtttc tgttttaaca acattttctg 6240 cgccgccaca aattttggct gcatcgacag ttttcttctg cccaattcca gaaacgaaga 6300 00E9 aatgatgggt gatggtttcc tttggtgcta ctgctgccgg tttgttttga acagtaaacg 6360 9877778777 09E9 tctgttgagc acatcctgta ataagcaggg ccagcgcagt agcgagtagc atttttttca 6420 9799 tggtgttatt cccgatgctt tttgaagttc gcagaatcgt atgtgtagaa aattaaacaa 6480 7787787887 accctaaaca atgagttgaa atttcatatt gttaatattt attaatgtat gtcaggtgcg 6540 atgaatcgtc attgtattcc cggattaact atgtccacag ccctgacggg gaacttctct 6600 0099 gcgggagtgt ccgggaataa ttaaaacgat gcacacaggg tttagcgcgt acacgtattg 6660 0999 the cattatgcca acgccccggt gctgacacgg aagaaaccgg acgttatgat ttagcgtgga 6720 0229 aagatttgtg tagtgttctg aatgctctca gtaaatagta atgaattatc aaaggtatag 6780 08/9 the taatatcttt tatgttcatg gatatttgta acccatcgga aaactcctgc tttagcaaga 6840 the ttttccctgt attgctgaaa tgtgatttct cttgatttca acctatcata ggacgtttct 6900 0069 ataagatgcg tgtttcttga gaatttaaca tttacaacct ttttaagtcc ttttattaac 6960 0969 credit acggtgttat cgttttctaa cacgatgtga atattatctg tggctagata gtaaatataa 7020 020L the tgtgagacgt tgtgacgttt tagttcagaa taaaacaatt cacagtctaa atcttttcgc 7080 080L the acttgatcga atatttcttt aaaaatggca acctgagcca ttggtaaaac cttccatgtg 7140 atacgagggc gcgtagtttg cattatcgtt tttatcgttt caatctggtc tgacctcctt 7200 0022

7787777878 gtgttttgtt gatgatttat gtcaaatatt aggaatgttt tcacttaata gtattggttg 7260 0972

cgtaacaaag tgcggtcctg ctggcattct ggagggaaat acaaccgaca gatgtatgta 7320 OZEL

aggccaacgt gctcaaatct tcatacagaa agatttgaag taatatttta accgctagat 7380 08EL

the gaagagcaag cgcatggagc gacaaaatga ataaagaaca atctgctgat gatccctccg 7440

tggatctgat tcgtgtaaaa aatatgctta atagcaccat ttctatgagt taccctgatg 7500 0052

ttgtaattgc atgtatagaa cataaggtgt ctctggaagc attcagagca attgaggcag 7560 09SL

cgttggtgaa gcacgataat aatatgaagg attattccct ggtggttgac tgatcaccat 7620 0292

aactgctaat cattcaaact atttagtctg tgacagagcc aacacgcagt ctgtcactgt 7680 089L

the caggaaagtg gtaaaactgc aactcaatta ctgcaatgcc ctcgtaatta agtgaattta 7740 DILL

caatatcgtc ctgttcggag ggaagaacgc gggatgttca ttcttcatca cttttaattg 7800 008L cheese atgtatatgc tctcttttct gacgttagtc tccgacggca ggcttcaatg acccaggctg 7860 098L

agaaattccc ggaccctttt tgctcaagag cgatgttaat ttgttcaatc atttggttag 7920 0262

the gaaagcggat gttgcgggtt gttgttctgc gggttctgtt cttcgttgac atgaggttgc 7980 086L

cccgtattca gtgtcgctga tttgtattgt ctgaagttgt ttttacgtta agttgatgca 8040 0708

gatcaattaa tacgatacct gcgtcataat tgattatttg acgtggtttg atggcctcca 8100 00t8

cgcacgttgt gatatgtaga tgataatcat tatcacttta cgggtccttt ccggtgatcc 8160 09t8

gacaggttac ggcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 8220

catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 8280 0878

gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 8340

ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 8400

ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg 8460 7978

ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 8520 0258

the gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 8580 tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 8640 tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 8640 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta 8700 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta 8700 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 8760 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 8760 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 8820 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 8820 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 8880 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgo 8880 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 8940 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 8940 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 9000 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 9000 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 9060 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 9060 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 9120 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 9120 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 9180 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 9180 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 9240 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 9240 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 9300 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 9300 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 9360 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 9360 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 9420 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 9420 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 9480 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 9480 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 9540 gccaacttac ttctgacaac gatcggagga ccgaaggage taaccgcttt tttgcacaac 9540 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 9600 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 9600 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 9660 aacgacgage gtgacaccao gatgcctgta gcaatggcaa caacgttgcg caaactatta 9660 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 9720 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 9720 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 9780 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 9780 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 9840 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 9840 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 9900 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 9900 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 9960 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 9960 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 10020 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 10020 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 10080 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 10080 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 10140 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 10140 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 10200 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 10200 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 10260 10260 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtago accgcctaca gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 10320 10320 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 10380 10380 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 10440 10440 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 10500 10500 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 10560 10560 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 10620 10620 ctttatagto ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 10680 10680 tcaggggggo ggagcctatg gaaaaacgco agcaacccgg cctttttacg gttcctggcc tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 10740 10740 ttttgctggc cttttgctca catgtcctgc aggcag ttttgctggc cttttgctca catgtcctgc aggcag 10776 10776

<210> 12 <210> 12 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 12 <400> 12 gctccagcta cggaaagtca gattactgga gg gctccagcta cggaaagtca gattactgga gg 32 32

<210> 13 <210> 13 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 13 <400> 13 cctccagtaa tctgactttd cgtagctgga gc cctccagtaa tctgactttc cgtagctgga gc 32 32

<210> 14 <210> 14 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 14 <400> 14 caggctcctc aagtggtact actcggtgca tg caggctcctc aagtggtact actcggtgca tg 32 32

<210> 15 <210> 15 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 15 <400> 15 catgcaccga gtagtaccac ttgaggagcc tg 32 catgcaccga gtagtaccac ttgaggagcc tg 32

<210> 16 <210> 16 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 16 <400> 16 agagaagttc ggcaatcttg ttactggtac tc 32 agagaagttc ggcaatcttg ttactggtac tc 32

<210> 17 <210> 17 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 17 <400> 17 gagtaccagt aacaagattg ccgaacttct ct 32 gagtaccagt aacaagattg ccgaacttct ct 32

<210> 18 <210> 18 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 18 <400> 18 tggaccctta acattaggca gcgatccgtt ta 32 tggaccctta acattaggca gcgatccgtt ta 32

<210> 19 <210> 19 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 19 <400> 19 taaacggatc gctgcctaat gttaagggtc ca 32 taaacggatc gctgcctaat gttaagggtc ca 32

<210> 20 <210> 20 <211> 30 <211> 30 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 20 <400> 20 cgatccgttt atgtatcctt agtcaggtgt 30 cgatccgttt atgtatcctt agtcaggtgt 30

<210> 21 <210> 21 <211> 30 <211> 30 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 21 <400> 21 acacctgact aaggatacat aaacggatcg 30 acacctgact aaggatacat aaacggatcg 30

<210> 22 <210> 22 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 22 <400> 22 tcaatgtcac gttctcctac ctagttggtt gg 32 tcaatgtcac gttctcctac ctagttggtt gg 32

<210> 23 <210> 23 <211> 32 <211> 32 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 23 <400> 23 ccaaccaact aggtaggaga acgtgacatt ga 32 ccaaccaact aggtaggaga acgtgacatt ga 32

<210> 24 <210> 24 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 56 <223> Target 56

<400> 24 <400> 24 catcaagttc agctcttcct 20 catcaagttc agctcttcct 20

<210> 25 <210> 25 <211> 20 <211> 20 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Target 150 <223> Target 150

<400> 25 <400> 25 gatcacaagc aactctggat 20 gatcacaagc aactctggat 20

Claims (20)

The claims defining the invention are as follows:

1. A lymphocyte comprising an antigen targeting receptor and a suppressed autophagy gene, wherein the antigen targeting receptor comprises a synthetic receptor.

2. A lymphocyte as defined in claim 1, comprising a nucleic acid encoding the antigen targeting receptor inserted within a locus of the autophagy gene to disrupt expression of the autophagy gene.

3. A lymphocyte as defined in the claim 2, wherein the nucleic acid encoding the antigen targeting receptor is inserted within the locus of the autophagy gene so that expression of the antigen targeting receptor is regulated by an endogenous promoter ofthe autophagy gene.

4. A lymphocyte as defined in any one of claims 1 to 3, wherein the nucleic acid encoding the antigen targeting receptor is inserted within an exon of the autophagy gene.

5. A lymphocyte as defined in any one of claims 1 to 3, wherein the nucleic acid encoding the antigen targeting receptor is inserted within an intron of the autophagy gene, optionally wherein the autophagy gene is ATG5 and the nucleic acid encoding the antigen targeting receptor is inserted within intron 2 of the ATG5 gene, and further optionally where the nucleic acid used to insert the antigen targeting receptor has the sequence of one of SEQ ID NO:8 or SEQ ID NO:9.

?0 6. A lymphocyte as defined in claim 1, wherein the autophagy gene is at a first locus of a genome of the lymphocyte and the lymphocyte comprises a nucleic acid encoding the antigen targeting receptor at a second locus of the genome, the first locus being different from the second locus.

7. A lymphocyte as defined in claim 6, wherein expression of the antigen targeting receptor is regulated by a heterologous promoter.

8. A lymphocyte as defined in any one of claims 1 to 7, wherein the autophagy gene is ablated, or wherein the autophagy gene is suppressed by RNAi.

9. A method of conducting immunotherapy comprising administering a lymphocyte as defined in any one of claims 1 to 8 to a subject.

10. A method of conducting immunotherapy using lymphocytes, the method comprising administering to a subject lymphocytes that have been modified to suppress an autophagy gene, optionally wherein the subject is a mammal, and optionally wherein the subject is a human.

11. A method of conducting immunotherapy using lymphocytes, the method comprising administering to a subject lymphocytes that have been modified to express an antigen targeting receptor and to suppress an autophagy gene.

12. A method of treating cancer, the method comprising a step of conducting immunotherapy as defined in any one of claims 9-11, wherein optionally the cancer is B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell lymphoma or other lymphoid malignancy, liver, pancreatic, brain, breast, ovarian, colorectal, acute myeloid leukemia (AML), multiple myeloma, lung, gastric, glioma, EGFR-positive solid tumor, glioblastoma, glioblastoma multiforme, stomach, nasopharyngeal, esophageal, prostate, neuroblastoma, hepatocellular, squamous cell lung, MSLN-positive solid tumor, non-small-cell lung (NSCLC), triple-negative breast cancer (TNBC), sarcoma, advanced solid tumor, renal cell, central nervous system, or an ROR1-positive malignancy; and optionally wherein the cancer is ovarian cancer, breast cancer, or lung cancer.

13. A method of making a lymphocyte for use in immunotherapy, the method comprising a step of modifying the lymphocyte to suppress an autophagy gene, the method further comprising modifying the lymphocyte to express a synthetic antigen targeting receptor.

?5 14. A method as defined in claim 13, wherein a gene editing method is used to insert a nucleic acid encoding the antigen targeting receptor at a locus of the autophagy gene, optionally wherein the gene editing method is used to insert the nucleic acid encoding the antigen targeting receptor so that expression of the antigen targeting receptor is regulated by an endogenous promoter of the autophagy gene.

15. A method as defined in claim 13, wherein the step of modifying the lymphocyte to express the antigen targeting receptor comprises inserting a nucleic acid encoding the antigen targeting receptor within a genome of the lymphocyte at a first locus, and wherein the step of modifying the lymphocyte to suppress an autophagy gene comprises knocking out the autophagy gene at a second locus of the genome, the second locus being different from the first locus.

16. A lymphocyte or method as defined in any one of claims 1 to 15, wherein: the lymphocyte comprises a lymphocyte with activity in killing tumor cells; the lymphocyte comprises a cytotoxic lymphocyte; or the lymphocyte comprises a T-cell, a natural killer (NK) cell, or a B-cell, optionally wherein the T-cell comprises an induced pluripotent stem cell, a CD34+ T cell, a CD4+ T-cell, a CD8+ T-cell, a Treg cell, a tissue-resident memory T cell (TRM), or a natural killer T-cell (NKT).

17. A lymphocyte or method as defined in any one of claims 1 to 16, wherein the autophagy gene is ULKI, ULK2, ULK3, FIP200, Vps34, Beclin-1, p150, UVRAG, ATGI, ATG4, ATG5, ATG7, ATG8, ATG9, ATGIO, ATG12, ATG13, ATG14L, ATG16L, ATG16LI, ATG18, VMPI, or GABARAP; and optionally wherein the autophagy gene is ATG5 or ATG14.

18. A lymphocyte or method as defined in any one of claims 1 to 17, wherein the antigen targeting receptor comprises a chimeric antigen receptor (CAR) or an engineered T cell receptor.

19. A lymphocyte or method as defined in any one of claims 1 to 18, wherein the antigen targeting receptor is specific for a tumor-specific antigen, optionally wherein the tumor-specific antigen comprises the folate receptor (FR), the a-folate receptor, the p-folate receptor, the y-folate receptor, CD19, CD20, CD133, CD138, CEA, Claudin 18.2, EGFR, EGFRvIII, EphA2, EpCAM, GD2, GPC3, HER2, MSLN, MG7, MUC1, NY-ESO-1, LMP1, prostate specific membrane antigen (PSMA), Fra, NKG2DI, BCMA, IL13Ralpha2, LeY, CD70, B7-H3, ROR1, or PSCA.

20. A lymphocyte or method as defined in any one of claims 1 to 19, wherein the antigen targeting receptor comprises a chimeric antigen receptor (CAR), and wherein the CAR is an a-folate receptor CAR, and wherein optionally the nucleotide construct used to insert the CAR in the lymphocyte has the nucleic acid sequence of either one of SEQ ID NO:8 or SEQ ID NO:9.